Analyte sensors employing multiple enzymes and methods associated therewith

ABSTRACT

Multiple enzymes may be present in the active area(s) of an electrochemical sensor to facilitate analysis of one or more analytes. The multiple enzymes may function independently to detect several analytes or in concert to detect a single analyte. One sensor configuration includes a first active area and a second active area, where the first active area has an oxidation-reduction potential that is sufficiently separated from the oxidation-reduction potential of the second active area to allow independent signal production. Some sensor configurations may have an active area overcoated with a multi-component membrane containing two or more different membrane polymers. Sensor configurations having multiple enzymes capable of interacting in concert include those in which a first enzyme converts an analyte into a first product and a second enzyme converts the first product into a second product, thereby generating a signal at a working electrode that is proportional to the analyte concentration.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority of U.S.Provisional Application 62/797,566 entitled “Analyte Sensors EmployingMultiple Enzymes and Methods Associated Therewith,” filed on Jan. 28,2019.

BACKGROUND

The detection of various analytes within an individual can sometimes bevital for monitoring the condition of their health and well-being.Deviation from normal analyte levels can often be indicative of anunderlying physiological condition, such as a metabolic condition orillness, or exposure to particular environmental conditions.

Any analyte may be suitable for a physiological analysis provided that asuitable chemistry can be identified for sensing the analyte. To thisend, amperometric sensors configured for assaying glucose in vivo havebeen developed and refined over recent years. Other analytes commonlysubject to physiological dysregulation that may similarly be desirableto monitor either ex vivo or in vivo include, but are not limited to,lactate, oxygen, pH, A1c, ketones, drug levels, and the like.

Analyte monitoring in an individual may take place periodically orcontinuously over a period of time. Periodic analyte monitoring may takeplace by withdrawing a sample of bodily fluid, such as blood, at settime intervals and analyzing ex vivo. Continuous analyte monitoring maybe conducted using one or more sensors that remain at least partiallyimplanted within a tissue of an individual, such as dermally,subcutaneously or intravenously, so that analyses may be conducted invivo. Implanted sensors may collect analyte data at any dictated rate,depending on an individual's particular health needs and/or previouslymeasured analyte levels.

Periodic, ex vivo analyte monitoring can be sufficient to determine thephysiological condition of many individuals. However, ex vivo analytemonitoring may be inconvenient or painful for some persons. Moreover,there is no way to recover lost data if an analyte measurement is notobtained at an appropriate time.

Continuous analyte monitoring with an in vivo implanted sensor may be amore desirable approach for individuals having severe analytedysregulation and/or rapidly fluctuating analyte levels, although it canalso be beneficial for other individuals as well. While continuousanalyte monitoring with an implanted sensor can be advantageous, thereare challenges associated with these types of measurements. Intravenousanalyte sensors have the advantage of providing analyte concentrationsdirectly from blood, but they are invasive and can sometimes be painfulfor an individual to wear, particularly over an extended period.Subcutaneous, interstitial, or dermal analyte sensors can often be lesspainful for an individual to wear and can provide sufficient measurementaccuracy in many cases.

In vivo analyte sensors typically are configured to analyze for a singleanalyte in order to provide specific analyses, often employing an enzymeto provide the analytical specificity. However, the physiologicalinterplay between various combinations of analytes can makemulti-analyte analyses desirable in certain instances as well. Atpresent, in vivo analysis of multiple analytes may necessitate using acorresponding number of analyte sensors configured for analyzing eachanalyte. This approach may be inconvenient due to the requirement for anindividual to wear multiple analyte sensors. In addition, multipleanalyte sensors may represent an unacceptable cost burden for anindividual or an insurance provider. There is also an increasedopportunity for one of the independent analyte sensors to fail duringsuch sensing protocols.

In vivo analyte sensors may also include a membrane disposed over atleast the implanted portion of the analyte sensor. In one aspect, themembrane may improve biocompatibility of the analyte sensor. In anotheraspect, the membrane may be permeable or semi-permeable to an analyte ofinterest and limit the overall analyte flux to the active area of theanalyte sensor. That is, the membrane may function as a mass transportlimiting membrane. Limiting analyte access to the active area of thesensor with a mass transport limiting membrane can aid in avoidingsensor overload (saturation), thereby improving detection performanceand accuracy. Such membranes may be highly specific toward limiting masstransport of a particular analyte, with other substances permeatingthrough the membrane at significantly different rates. The differingmembrane permeability of various potential analytes represents asignificant hurdle for developing analyte sensors configured forassaying multiple analytes. Namely, the differing membrane permeabilityvalues may lead to significantly different sensitivities for themultiple analytes, thereby complicating analyses. The differingsensitivities for multiple analytes may sometimes be partiallycompensated for by using active areas of different sizes (e.g., smalleractive areas for analytes having high sensitivity/permeability andlarger active areas for analytes having lower sensitivity/permeability),but this approach may present significant manufacturing challenges andmay not be applicable in all cases.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are included to illustrate certain aspects of thepresent disclosure, and should not be viewed as exclusive embodiments.The subject matter disclosed is capable of considerable modifications,alterations, combinations, and equivalents in form and function, withoutdeparting from the scope of this disclosure.

FIG. 1 shows a diagram of an illustrative sensing system that mayincorporate an analyte sensor of the present disclosure.

FIG. 2A shows a diagram of an illustrative two-electrode analyte sensorconfiguration having a single working electrode, which is compatible foruse in some embodiments of the disclosure herein. FIGS. 2B and 2C showdiagrams of illustrative three-electrode analyte sensor configurationshaving a single working electrode, which are compatible for use in someembodiments of the disclosure herein.

FIG. 3 shows a diagram of an illustrative analyte sensor configurationhaving two working electrodes, a reference electrode and a counterelectrode, which is compatible for use in some embodiments of thedisclosure herein.

FIG. 4 shows an illustrative analyte sensor configuration compatible foruse in some embodiments of the disclosure herein, in which two differentactive areas are disposed upon the surface of a single workingelectrode.

FIG. 5A shows the concerted enzymatic reaction cycle associated withethanol detection using alcohol oxidase and xanthine oxidase locateddirectly upon a working electrode, according to various embodiments ofthe present disclosure.

FIG. 5B shows the concerted enzymatic reaction cycle associated withketone detection using β-hydroxybutyrate dehydrogenase, nicotinamideadenine dinucleotide, and diaphorase located directly upon a workingelectrode, according to various embodiments of the present disclosure.

FIG. 5C shows the concerted enzymatic reaction cycle associated withketone detection using β-hydroxybutyrate dehydrogenase, nicotinamideadenine dinucleotide, NADH oxidase, and superoxide dismutase locateddirectly upon a working electrode, according to various embodiments ofthe present disclosure

FIG. 5D shows the concerted enzymatic reaction cycle associated withketone detection using β-hydroxybutyrate dehydrogenase, nicotinamideadenine dinucleotide, and poly-1,10-phenanthroline-5,6-dione locateddirectly upon a working electrode, according to various embodiments ofthe present disclosure.

FIG. 5E shows the concerted enzymatic reaction cycle associated withethanol detection using glucose oxidase, catalase, and xanthine oxidase,in which glucose oxidase is remote from a working electrode and xanthineoxidase is located directly upon the working electrode, according tovarious embodiments of the present disclosure.

FIGS. 6A and 6B show diagrams of illustrative working electrodes inwhich a first active area is disposed directly upon a surface of theworking electrode and a second active area is separated from the workingelectrode by a membrane, and which is compatible for use in someembodiments of the disclosure herein. FIG. 6C shows a diagram anillustrative working electrode in which a first active area and a secondactive area are laterally spaced apart from one another upon a workingelectrode, and one of the active areas is separated from the workingelectrode by a membrane.

FIG. 7 shows an illustrative schematic of a portion of an analyte sensorhaving two working electrodes and featuring a bilayer membraneovercoating one of the two working electrodes, which is compatible foruse in some embodiments of the disclosure herein.

FIG. 8 shows an illustrative cyclic voltammogram obtained for ananalyte-free buffer solution using a working electrode containing twodifferent osmium complexes as electron transfer mediators. FIG. 9 showsfour replicates of the electrode response in a 5 mM glucose/5 mM lactatebuffer when cycling the electrode of FIG. 8 between the E1 and E2potentials.

FIG. 10 shows three replicates of the response for an electrodecontaining alcohol oxidase and xanthine oxidase together in an activearea upon exposure to varying ethanol concentrations.

FIG. 11A shows an illustrative plot of average current response versusethanol concentration for the electrodes of FIG. 10. FIG. 11B shows anillustrative plot of current response versus ethanol concentration for asingle electrode.

FIG. 12A shows two replicates of the response for an electrodecontaining glucose oxidase and xanthine oxidase layered in separateactive areas and spaced apart by a membrane upon exposure to varyingethanol concentrations, in which catalase is present in the active areacontaining glucose oxidase. FIG. 12B shows comparative response databetween an electrode containing glucose oxidase and xanthine oxidaselayered in separate active areas and spaced apart by a membrane uponexposure to varying ethanol concentrations, in which catalase is presentin the active areas separately.

FIG. 13 shows an illustrative plot of average current response versusethanol concentration for the electrodes of FIG. 12A.

FIG. 14 shows an illustrative plot of the response of an electrodeovercoated with Polymers 1A and 1B to a 5 mM lactate solution.

FIG. 15 shows an illustrative plot of the response of an electrodeovercoated with Polymer 2 to a 5 mM lactate solution.

FIG. 16 shows an illustrative plot of the response of an electrodeovercoated with Polymer 3 to a 5 mM lactate solution.

FIG. 17 shows an illustrative plot of the response of an electrodeovercoated with Polymer 3 to lactate solutions having varying lactateconcentrations.

FIG. 18 shows an illustrative plot of the response of an electrodeovercoated with Polymer 4 to a 5 mM lactate solution.

FIG. 19 shows an illustrative plot of the response of an electrodeovercoated with Polymer 4 to lactate solutions having varying lactateconcentrations.

FIG. 20 shows an illustrative plot of the response of an electrodeovercoated with a bilayer membrane comprising a lower layer ofcrosslinked PVP (Polymer 2) and an upper layer of crosslinked Polymer 1Bto a 5 mM lactate solution.

FIG. 21 shows an illustrative plot of the response of an electrodeovercoated with a bilayer membrane comprising a lower layer ofcrosslinked PVP (Polymer 2) and an upper layer of crosslinked Polymer 1Ato a 5 mM lactate solution, in which the electrode was dip coated avariable number of times with Formulation 1 and Formulation 3.

FIG. 22 shows an illustrative plot of the response of an electrodeovercoated with an admixed membrane comprising crosslinked PVP (Polymer2) and crosslinked Polymer 1B to a 5 mM lactate solution.

FIG. 23 shows an illustrative plot of the response of an electrodeovercoated with an admixed membrane comprising crosslinked PVP (Polymer2) and crosslinked Polymer 1B to varying lactate concentrations.

FIG. 24 shows an illustrative plot of the response an electrodeovercoated with an admixed membrane comprising various ratios ofcrosslinked PVP (Polymer 2) and crosslinked Polymer 1B.

FIG. 25 shows an illustrative plot of the response of a sensorcontaining two working electrodes to a 30 mM glucose/5 mM lactatesolution, in which the lactate-responsive working electrode isovercoated with a bilayer membrane and the glucose-responsive workingelectrode is overcoated with a homogeneous membrane.

FIG. 26 shows an illustrative plot of the response of a sensorcontaining two working electrodes to solutions containing varyingconcentrations of glucose and lactate, in which the lactate-responsiveworking electrode is overcoated with a bilayer membrane and theglucose-responsive working electrode is overcoated with a homogeneousmembrane.

FIG. 27 shows four replicates of the response for an electrodecontaining diaphorase, NAD⁺, and β-hydroxybutyrate dehydrogenase whenexposed to varying β-hydroxybutyrate concentrations.

FIG. 28 shows an illustrative plot of average current response versusβ-hydroxybutyrate concentration for the electrodes of FIG. 27.

FIG. 29 shows an illustrative plot of current response for theelectrodes of FIG. 27 when exposed to 8 mM of β-hydroxybutyrate in 100mM PBS at 33° C. for 2 weeks.

FIG. 30 shows an illustrative plot of sensor performance for Groups 1-4in Example 6.

FIG. 31 shows an illustrative plot of the response of a Group 1 analytesensor from Example 6 to lactate solutions having varying lactateconcentrations.

FIG. 32 is a schematic diagram of an example analyte monitoring andvehicle control system, according to one or more embodiments of thepresent disclosure.

DETAILED DESCRIPTION

The present disclosure generally describes analyte sensors and methodsemploying multiple enzymes for detection and, more specifically, analytesensors and methods in which multiple enzymes may function independentlyor in concert to detect one or more analytes.

As discussed above, analyte sensors are commonly used to detect a singleanalyte. If detection of multiple analytes is desired, a correspondingnumber of analyte sensors may be employed. This approach may beundesirable due to, among other things, cost concerns, the necessity foran individual to wear multiple analyte sensors, and an increasedlikelihood of individual sensor failure.

Some analyte sensors utilize an enzymatic reaction as the basis fordetecting an analyte of interest. Since enzymes often display reactionspecificity toward a particular substrate or related class ofsubstrates, they may provide analyte sensors having detection chemistryconfigured to analyze for a single analyte of interest. As such, analytesensors for analyzing a single analyte usually incorporate only acorresponding single enzyme to promote a suitable enzymatic reaction forfacilitating detection. At present, it can be rather difficult toincorporate multiple enzymes in an analyte sensor in order to providedetection capabilities for multiple analytes. Reasons includedifferences in analyte sensitivity and potential incompatibility of oneor more of the enzymes to a given set of analysis conditions.

In contrast to analyte sensors featuring a single enzyme, the presentdisclosure describes analyte sensors in which multiple enzymes arepresent in the active area(s) of the sensors. A number of advantages maybe realized by incorporating multiple enzymes in an analyte sensor inthe various manners described herein. In some sensor configurations ofthe present disclosure, the multiple enzymes may facilitate independentdetection of multiple analytes, such as glucose and lactate. Membranesconfigured to provide tailored permeability for multiple analytes arealso described herein, which may facilitate analyte detection with asingle analyte sensor by levelizing the sensor's sensitivity toward eachanalyte. In other sensor configurations of the present disclosure,multiple enzymes may be chosen to function in concert to facilitatedetection of a single analyte of interest, which may otherwise beproblematic or impossible to assay using a single enzyme. In any event,fewer electrodes may be needed to detect a given analyte or set ofanalytes than would otherwise be feasible. Moreover, the presentdisclosure may afford sensors having a reduced size and a decreasedcomplexity of their measurement electronics than would otherwise bepossible. Thus, analyte sensors employing multiple enzymes in variousconfigurations may facilitate efficient detection of one or moreanalytes according to the disclosure herein.

Analyte sensors containing multiple enzymes, whether operatingindependently or in concert, may function with enhanced stability in thepresence of an appropriate stabilizer. Stabilizers that may be usedinclude, for example, catalase or albumin (e.g., bovine serum albumin orhuman serum albumin). Catalase is known for its ability to clearreactive species, such as peroxide, from biological environments.Albumins, in contrast, are not believed to exhibit functionality toclear reactive species, and their ability to stabilize the response ofthe analyte sensors of the present disclosure is surprising as a result.

Before describing the analyte sensors of the present disclosure in moredetail, a brief overview of suitable in vivo analyte sensorconfigurations and sensor systems employing the analyte sensors willfirst be provided so that the embodiments of the present disclosure maybe better understood. It is to be understood that any of the sensorsystems and analyte sensor configurations described hereinafter mayfeature multiple enzymes, in accordance with the various embodiments ofthe present disclosure.

FIG. 1 shows a diagram of an illustrative sensing system that mayincorporate an analyte sensor of the present disclosure. As shown,sensing system 100 includes sensor control device 102 and reader device120 that are configured to communicate with one another over a localcommunication path or link, which may be wired or wireless, uni- orbi-directional, and encrypted or non-encrypted. Reader device 120 mayconstitute an output medium for viewing analyte concentrations andalerts or notifications determined by sensor 104 or a processorassociated therewith, as well as allowing for one or more user inputs,according to some embodiments. Reader device 120 may be a multi-purposesmartphone or a dedicated electronic reader instrument. While only onereader device 120 is shown, multiple reader devices 120 may be presentin certain instances. Reader device 120 may also be in communicationwith remote terminal 170 and/or trusted computer system 180 viacommunication path(s)/link(s) 141 and/or 142, respectively, which alsomay be wired or wireless, uni- or bi-directional, and encrypted ornon-encrypted. Reader device 120 may also or alternately be incommunication with network 150 (e.g., a mobile telephone network, theinternet, or a cloud server) via communication path/link 151. Network150 may be further communicatively coupled to remote terminal 170 viacommunication path/link 152 and/or trusted computer system 180 viacommunication path/link 153. Alternately, sensor 104 may communicatedirectly with remote terminal 170 and/or trusted computer systems 180without an intervening reader device 120 being present. For example,sensor 104 may communicate with remote terminal 170 and/or trustedcomputer system 180 through a direct communication link to network 150,according to some embodiments, as described in U.S. Patent ApplicationPublication 2011/0213225 an incorporated herein by reference in itsentirety. Any suitable electronic communication protocol may be used foreach of the communication paths or links, such as near fieldcommunication (NFC), radio frequency identification (RFID), BLUETOOTH®or BLUETOOTH® Low Energy protocols, WiFi, or the like. Remote terminal170 and/or trusted computer system 180 may be accessible, according tosome embodiments, by individuals other than a primary user who have aninterest in the user's analyte levels. Reader device 120 may comprisedisplay 122 and optional input component 121. Display 122 may comprise atouch-screen interface, according to some embodiments.

Sensor control device 102 includes sensor housing 103, which may housecircuitry and a power source for operating sensor 104. Optionally, thepower source and/or active circuitry may be omitted. A processor (notshown) may be communicatively coupled to sensor 104, with the processorbeing physically located within sensor housing 103 or reader device 120.Sensor 104 protrudes from the underside of sensor housing 103 andextends through adhesive layer 105, which is adapted for adhering sensorhousing 103 to a tissue surface, such as skin, according to someembodiments.

Sensor 104 is adapted to be at least partially inserted into a tissue ofinterest, such as within the dermal or subcutaneous layer of the skin.Sensor 104 may comprise a sensor tail of sufficient length for insertionto a desired depth in a given tissue. The sensor tail may comprise atleast one working electrode and one or more active areas (sensingregions/spots or sensing layers) located upon the at least one workingelectrode and that are active for sensing one or more analytes ofinterest. Collectively, the one or more active areas may comprisemultiple enzymes, according to one or more embodiments of the presentdisclosure. The active areas may include a polymeric material to whichat least some of the enzymes are covalently bonded, according to someembodiments. In various embodiments of the present disclosure, analytesmay be monitored in any biological fluid of interest such as dermalfluid, interstitial fluid, plasma, blood, lymph, synovial fluid,cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, orthe like. In particular embodiments, analyte sensors of the presentdisclosure may be adapted for assaying dermal fluid or interstitialfluid.

In some embodiments, sensor 104 may automatically forward data to readerdevice 120. For example, analyte concentration data may be communicatedautomatically and periodically, such as at a certain frequency as datais obtained or after a certain time period has passed, with the databeing stored in a memory until transmittal (e.g., every minute, fiveminutes, or other predetermined time period). In other embodiments,sensor 104 may communicate with reader device 120 in a non-automaticmanner and not according to a set schedule. For example, data may becommunicated from sensor 104 using RFID technology when the sensorelectronics are brought into communication range of reader device 120.Until communicated to reader device 120, data may remain stored in amemory of sensor 104. Thus, a patient does not have to maintain closeproximity to reader device 120 at all times, and can instead upload dataat a convenient time. In yet other embodiments, a combination ofautomatic and non-automatic data transfer may be implemented. Forexample, data transfer may continue on an automatic basis until readerdevice 120 is no longer in communication range of sensor 104.

An introducer may be present transiently to promote introduction ofsensor 104 into a tissue. In illustrative embodiments, the introducermay comprise a needle or similar sharp. It is to be recognized thatother types of introducers, such as sheaths or blades, may be present inalternative embodiments. More specifically, the needle or otherintroducer may transiently reside in proximity to sensor 104 prior totissue insertion and then be withdrawn afterward. While present, theneedle or other introducer may facilitate insertion of sensor 104 into atissue by opening an access pathway for sensor 104 to follow. Forexample, the needle may facilitate penetration of the epidermis as anaccess pathway to the dermis to allow implantation of sensor 104 to takeplace, according to one or more embodiments. After opening the accesspathway, the needle or other introducer may be withdrawn so that it doesnot represent a sharps hazard. In illustrative embodiments, suitableneedles may be solid or hollow, beveled or non-beveled, and/or circularor non-circular in cross-section. In more particular embodiments,suitable needles may be comparable in cross-sectional diameter and/ortip design to an acupuncture needle, which may have a cross-sectionaldiameter of about 250 microns. It is to be recognized, however, thatsuitable needles may have a larger or smaller cross-sectional diameterif needed for particular applications.

In some embodiments, a tip of the needle (while present) may be angledover the terminus of sensor 104, such that the needle penetrates atissue first and opens an access pathway for sensor 104. In otherillustrative embodiments, sensor 104 may reside within a lumen or grooveof the needle, with the needle similarly opening an access pathway forsensor 104. In either case, the needle is subsequently withdrawn afterfacilitating sensor insertion.

The analyte sensors described herein may feature multiple enzymes uponthe active area(s) of a single working electrode or upon two or moreseparate working electrodes. Single working electrode configurations foran analyte sensor may employ two-electrode or three-electrode detectionmotifs, according to various embodiments of the present disclosure.Sensor configurations featuring a single working electrode are describedhereinafter in reference to FIGS. 2A-2C. Sensor configurations featuringmultiple working electrodes are described separately thereafter inreference to FIG. 3. Multiple enzymes may be incorporated in any of thesensor configurations described hereinafter, with specificconfigurations suitable for incorporating the multiple enzymes beingdescribed in further detail hereinbelow.

When a single working electrode is present in an analyte sensor,three-electrode detection motifs may comprise a working electrode, acounter electrode, and a reference electrode. Related two-electrodedetection motifs may comprise a working electrode and a secondelectrode, in which the second electrode functions as both a counterelectrode and a reference electrode (i.e., a counter/referenceelectrode). In both two-electrode and three-electrode detection motifs,one or more active areas of the analyte sensor may be in contact withthe working electrode. The one or more active areas may comprisemultiple enzymes according to the embodiments of the present disclosure,with the multiple enzymes being present in a single active area and/orin multiple active areas. In some embodiments, the various electrodesmay be at least partially stacked (layered) upon one another, asdescribed in further detail hereinafter. In some or other embodiments,the various electrodes may be laterally spaced apart from one anotherupon the sensor tail. Similarly, the associated active areas upon eachelectrode may be stacked vertically upon top of one another or belaterally spaced apart. In either case, the various electrodes may beelectrically isolated from one another by a dielectric material orsimilar insulator.

FIG. 2A shows a diagram of an illustrative two-electrode analyte sensorconfiguration having a single working electrode, which is compatible foruse in some embodiments of the disclosure herein. As shown, analytesensor 200 comprises substrate 212 disposed between working electrode214 and counter/reference electrode 216. Alternately, working electrode214 and counter/reference electrode 216 may be located upon the sameside of substrate 212 with a dielectric material interposed in between(configuration not shown). Active area 218 is disposed as at least onelayer upon at least a portion of working electrode 214. In variousembodiments, active area 218 may comprise multiple spots or a singlespot configured for detection of one or more analytes of interest.Collectively, multiple enzymes may be present in active area 218 (i.e.,in a single spot or in multiple spots).

Referring still to FIG. 2A, membrane 220 overcoats at least active area218 and may optionally overcoat some or all of working electrode 214and/or counter/reference electrode 216, or the entirety of analytesensor 200, according to some embodiments. One or both faces of analytesensor 200 may be overcoated with membrane 220. Membrane 220 maycomprise one or more polymeric membrane materials having capabilities oflimiting analyte flux to active area 218. Depending on the identity ofthe analyte(s), the composition of membrane 220 may vary, as describedfurther herein. Analyte sensor 200 may be operable for assaying the oneor more analytes by any of coulometric, amperometric, voltammetric, orpotentiometric electrochemical detection techniques.

FIGS. 2B and 2C show diagrams of illustrative three-electrode analytesensor configurations having a single working electrode, which arecompatible for use in some embodiments of the disclosure herein.Three-electrode analyte sensor configurations employing a single workingelectrode may be similar to that shown for analyte sensor 200 in FIG.2A, except for the inclusion of additional electrode 217 in analytesensors 201 and 202 (FIGS. 2B and 2C). With additional electrode 217,counter/reference electrode 216 may then function as either a counterelectrode or a reference electrode, and additional electrode 217fulfills the other electrode function not otherwise accounted for.Working electrode 214 continues to fulfill its original function.Additional electrode 217 may be disposed upon either working electrode214 or electrode 216, with a separating layer of dielectric material inbetween. For example, as depicted in FIG. 2B, dielectric layers 219 a,219 b and 219 c separate electrodes 214, 216 and 217 from one anotherand provide electrical isolation. Alternately, at least one ofelectrodes 214, 216 and 217 may be located upon opposite faces ofsubstrate 212, as shown in FIG. 2C. Thus, in some embodiments, electrode214 (working electrode) and electrode 216 (counter electrode) may belocated upon opposite faces of substrate 212, with electrode 217(reference electrode) being located upon one of electrodes 214 or 216and spaced apart therefrom with a dielectric material. Referencematerial layer 230 (e.g., Ag/AgCl) may be present upon electrode 217,with the location of reference material layer 230 not being limited tothat depicted in FIGS. 2B and 2C. As with sensor 200 shown in FIG. 2A,active area 218 in analyte sensors 201 and 202 may comprise multiplespots or a single spot configured for detection of one or more analytesof interest. Collectively, multiple enzymes may be present in activearea 218 of analyte sensors 201 and 202. Additionally, analyte sensors201 and 202 may be operable for assaying the one or more analytes by anyof coulometric, amperometric, voltammetric, or potentiometricelectrochemical detection techniques.

Like analyte sensor 200, membrane 220 may also overcoat active area 218,as well as other sensor components, in analyte sensors 201 and 202.Additional electrode 217 may be overcoated with membrane 220 in someembodiments. Although FIGS. 2B and 2C have depicted all of electrodes214, 216 and 217 as being overcoated with membrane 220, it is to berecognized that only working electrode 214 may be overcoated in someembodiments. Moreover, the thickness of membrane 220 at each ofelectrodes 214, 216 and 217 may be the same or different. As intwo-electrode analyte sensor configurations (FIG. 2A), one or both facesof analyte sensors 201 and 202 may be overcoated with membrane 220 inthe sensor configurations of FIGS. 2B and 2C, or the entirety of analytesensors 201 and 202 may be overcoated. Accordingly, the three-electrodesensor configurations shown in FIGS. 2B and 2C should be understood asbeing non-limiting of the embodiments disclosed herein, with alternativeelectrode and/or layer configurations remaining within the scope of thepresent disclosure.

Analyte sensor configurations having multiple working electrodes willnow be described in further detail. Although the following descriptionis primarily directed to analyte sensor configurations having twoworking electrodes, it is to be appreciated that more than two workingelectrodes may be successfully incorporated through an extension of thedisclosure herein. Additional working electrodes may allow additionalactive area(s) and corresponding sensing capabilities to be imparted toanalyte sensors having such features.

FIG. 3 shows a diagram of an illustrative analyte sensor configurationhaving two working electrodes, a reference electrode and a counterelectrode, which is compatible for use in some embodiments of thedisclosure herein. As shown in FIG. 3, analyte sensor 300 includesworking electrodes 304 and 306 disposed upon opposite faces of substrate302. Active area 310 is disposed upon the surface of working electrode304, and active area 312 is disposed upon the surface of workingelectrode 306. Collectively, multiple enzymes may be present in activeareas 310 and 312, with each active area 310,312 containing one or moreenzymes. For example, a glucose-responsive enzyme may be present inactive area 310 and a lactate-responsive enzyme may be present in activearea 312 in particular embodiments. Counter electrode 320 iselectrically isolated from working electrode 304 by dielectric layer322, and reference electrode 321 is electrically isolated from workingelectrode 306 by dielectric layer 323. Outer dielectric layers 330 and332 are positioned upon reference electrode 321 and counter electrode320, respectively. Membrane 340 may overcoat at least active areas 310and 312, according to various embodiments. Other components of analytesensor 300 may be overcoated with membrane 340 as well, and as above,one or both faces of analyte sensor 300, or a portion thereof, may beovercoated with membrane 340. Like analyte sensors 200, 201 and 202,analyte sensor 300 may be operable for assaying one or more analytes byany of coulometric, amperometric, voltammetric, or potentiometricelectrochemical detection techniques.

Alternative analyte sensor configurations having multiple workingelectrodes and differing from that shown in FIG. 3 may feature acounter/reference electrode instead of separate counter and referenceelectrodes 320,321, and/or feature layer and/or membrane arrangementsvarying from those expressly depicted. For example, the positioning ofcounter electrode 320 and reference electrode 321 may be reversed fromthat depicted in FIG. 3. In addition, working electrodes 304 and 306need not necessarily reside upon opposing faces of substrate 302 in themanner shown in FIG. 3.

Analyte sensor configurations featuring a working electrode having anactive area remote therefrom are shown in FIGS. 6A and 6B and discussedfurther below.

According to various embodiments of the present disclosure, an electrontransfer agent may be present in one or more of the active areas of anyof the analyte sensors or analyte sensor configurations disclosedherein. Suitable electron transfer agents may facilitate conveyance ofelectrons to the working electrode when an analyte (enzyme substrate)undergoes an oxidation-reduction reaction. Choice of the electrontransfer agent within each active area may dictate theoxidation-reduction potential observed for each. When multiple activeareas are present, the electron transfer agent within each active areamay be the same or different.

Suitable electron transfer agents may include electroreducible andelectrooxidizable ions, complexes or molecules (e.g., quinones) havingoxidation-reduction potentials that are a few hundred millivolts aboveor below the oxidation-reduction potential of the standard calomelelectrode (SCE). According to some embodiments, suitable electrontransfer agents may include low-potential osmium complexes, such asthose described in U.S. Pat. Nos. 6,134,461 and 6,605,200, which areincorporated herein by reference in their entirety. Additional examplesinclude those described in U.S. Pat. Nos. 6,736,957, 7,501,053 and7,754,093, the disclosures of each of which are incorporated herein byreference in their entirety. Other suitable electron transfer agents maycomprise metal compounds or complexes of ruthenium, osmium, iron (e.g.,polyvinylferrocene or hexacyanoferrate), or cobalt, includingmetallocene compounds thereof, for example. Suitable examples ofelectron transfer mediators and polymer-bound electron transfermediators may include those described in U.S. Pat. Nos. 8,444,834,8,268,143 and 6,605,201, the disclosures of which are incorporatedherein by reference in their entirety. Suitable ligands for the metalcomplexes may also include, for example, bidentate or higher denticityligands such as, for example, bipyridine, biimidazole, phenanthroline,or pyridyl(imidazole). Other suitable bidentate ligands may include, forexample, amino acids, oxalic acid, acetylacetone, diaminoalkanes, oro-diaminoarenes. Any combination of monodentate, bidentate, tridentate,tetradentate, or higher denticity ligands may be present in a metalcomplex to achieve a full coordination sphere.

According to various embodiments of the present disclosure, a polymermay be present in each active area of any of the analyte sensors oranalyte sensor configurations disclosed herein. Suitable polymers forinclusion in the active areas may include, but are not limited to,polyvinylpyridines (e.g., poly(4-vinylpyridine)), polyvinylimidazoles(e.g., poly(1-vinylimidazole)), or any copolymer thereof. Illustrativecopolymers that may be suitable for inclusion in the active areasinclude those containing monomer units such as styrene, acrylamide,methacrylamide, or acrylonitrile, for example. When multiple activeareas are present, the polymer within each active area may be the sameor different.

According to various embodiments of the present disclosure, the electrontransfer agent may be covalently bonded to the polymer in each activearea. The manner of covalent bonding is not considered to beparticularly limited. Covalent bonding of the electron transfer agent tothe polymer may take place by polymerizing a monomer unit bearing acovalently bound electron transfer agent, or the electron transfer agentmay be reacted with the polymer separately after the polymer has alreadybeen synthesized. According to some embodiments, a bifunctional spacermay covalently bond the electron transfer agent to the polymer withinthe active area, with a first functional group being reactive with thepolymer (e.g., a functional group capable of quaternizing a pyridinenitrogen atom or an imidazole nitrogen atom) and a second functionalgroup being reactive with the electron transfer agent (e.g., afunctional group that is reactive with a ligand coordinating a metalion).

Similarly, according to some or other various embodiments of the presentdisclosure, the enzyme within one or more of the active areas may becovalently bonded to the polymer. When multiple enzymes are present in asingle active area, all of the multiple enzymes may be covalently bondedto the polymer in some embodiments, and in other embodiments, only aportion of the multiple enzymes may be covalently bonded to the polymer.For example, a first enzyme may be covalently bonded to the polymer anda second enzyme may be non-covalently associated with the polymer.According to more specific embodiments, covalent bonding of the enzymeto the polymer may take place via a crosslinker introduced with asuitable crosslinking agent. Suitable crosslinking agents for reactionwith free amino groups in the enzyme (e.g., with the free amine inlysine) may include crosslinking agents such as, for example,polyethylene glycol diglycidylether (PEGDGE) or other polyepoxides,cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin,or derivatized variants thereof. Suitable crosslinking agents forreaction with free carboxylic acid groups in the enzyme may include, forexample, carbodiimides. The crosslinking is generally intermolecular,but can be intramolecular in some embodiments.

The electron transfer agent and/or the enzyme may be associated with thepolymer in the active area through means other than covalent bonding aswell. In some embodiments, the electron transfer agent and/or the enzymemay be ionically or coordinatively associated with the polymer. Forexample, a charged polymer may be ionically associated with anoppositely charged electron transfer agent or enzyme. In still otherembodiments, the electron transfer agent and/or the enzyme may bephysically entrained within the polymer without being bonded thereto.

Various configurations suitable for arranging multiple enzymes inanalyte sensors of the present disclosure will now be described infurther detail. The multiple enzymes may be deposited within one or moreactive areas of the sensors. The active areas may range in size fromabout 0.01 mm² to about 1 mm², although larger or smaller active areasare also contemplated herein.

In some embodiments, multiple enzymes may be arranged within separateactive areas upon a single working electrode. When the multiple enzymesare arranged in this manner, each active area may facilitate detectionof separate analytes, as described hereinafter. At least one of theactive areas may produce a signal independently of the other activeareas.

According to some embodiments, analyte sensors of the present disclosurehaving multiple active areas upon a single working electrode maycomprise: a sensor tail comprising at least a working electrode, and atleast two active areas disposed upon a surface of the working electrode.Each active area comprises an analyte-responsive enzyme and a polymer,with the analyte-responsive enzyme in each active area being different.Each active area has an oxidation-reduction potential, and theoxidation-reduction potential of a first active area is sufficientlyseparated from the oxidation-reduction potential of a second active areato allow production of a signal from the first active area independentof a signal from the second active area. In more specific embodiments,such analyte sensors may comprise a single working electrode having theat least two active areas. An electron-transfer agent may beincorporated within each active area to promote electron transfer.

Alternative sensor configurations may comprise a single active areacontaining both the first analyte-responsive enzyme and the secondanalyte-responsive enzyme, along with an electron transfer agent. Eachenzyme may be covalently bonded to separate portions of the polymer inthe single active area. Provided that the sensing chemistries forpromoting electron transfer for each analyte are not overly diluted inthe single active area, the single active area may facilitate analytedetection in a manner similar to that described below for separateactive areas. Such sensor configurations may be particularly feasiblewhen the analytes to be assayed with the first and secondanalyte-responsive enzymes have comparable membrane permeability values.

In more specific embodiments, the sensor tail may be configured forinsertion into a tissue. Suitable tissues are not considered to beparticularly limited and are addressed in more detail above. Similarly,considerations for deploying a sensor tail at a particular positionwithin a tissue are addressed above.

In more specific embodiments, the oxidation-reduction potentialassociated with the first active area may be separated from theoxidation-reduction potential of the second active area by at leastabout 100 mV, or by at least about 150 mV, or by at least about 200 mV.The upper limit of the separation between the oxidation-reductionpotentials is dictated by the working electrochemical window in vivo. Byhaving the oxidation-reduction potentials of the active areassufficiently separated in magnitude from one another, an electrochemicalreaction may take place within the first active area withoutsubstantially inducing an electrochemical reaction within the secondactive area. Thus, a signal from the first active area may beindependently produced at or above its corresponding oxidation-reductionpotential. At or above the oxidation-reduction potential of the secondactive area, in contrast, electrochemical reactions may occur withinboth active areas. As such, the resulting signal at or above theoxidation-reduction potential of the second active area may include asignal contribution from both the first active area and the secondactive area, and the signal is a composite signal. The signalcontribution from the second active area at or above itsoxidation-reduction potential may then be determined by subtracting fromthe composite signal the signal obtained solely from the first activearea at or above its corresponding oxidation-reduction potential.Similar considerations apply to analyzing the signal contributions froma single active area containing two different enzymes that producesignals at different oxidation-reduction potentials.

In more specific embodiments, the first and second active areas maycontain different electron transfer agents when the active areas arelocated upon the same working electrode, so as to affordoxidation-reduction potentials that are sufficiently separated inmagnitude. More specifically, the first active area may comprise a firstelectron transfer agent and the second active area may comprise a secondelectron transfer agent, with the first and second electron transferagents being different. The metal center and/or the ligands present in agiven electron transfer agent may be varied to provide sufficientseparation of the oxidation-reduction potentials of the first and secondactive areas, according to various embodiments of the presentdisclosure. According to still more specific embodiments, the firstelectron transfer agent may be covalently bonded to the polymer in thefirst active area, and the second electron transfer agent may becovalently bonded to the polymer in the second active area. The mannerof covalent bonding for the first electron transfer agent and the secondelectron transfer agent may be the same or different. Similarconsiderations apply to choosing electron transfer agents suitable foruse in conjunction with a first analyte-responsive enzyme and a secondanalyte-responsive enzyme contained within a single active area, inaccordance with the disclosure above.

In more specific embodiments of the present disclosure, theanalyte-responsive enzyme in each active area may be covalently bonded(or otherwise immobilized) to the polymer within each active area. Instill more specific embodiments, the analyte-responsive enzyme and theelectron transfer agent in each active area may be covalently bonded tothe polymer within each active area. When contained in a single activearea, the first analyte-responsive enzyme and a first electron transferagent may be covalently bonded to a first portion of polymer, and thesecond analyte-responsive enzyme and a second electron transfer agentmay be covalently bonded to a second portion of polymer. The polymer inthe first portion and the second portion may be the same or different.

Ideally, first and second active areas located upon a single workingelectrode may be configured to attain a steady state current rapidlyupon operating the analyte sensor at a given potential. Rapid attainmentof a steady state current may be promoted by choosing an electrontransfer agent for each active area that changes its oxidation statequickly upon being exposed to a potential at or above itsoxidation-reduction potential. Making the active areas as thin aspossible may also facilitate rapid attainment of a steady state current.For example, suitable thicknesses for the first and second active areasmay range from about 0.1 microns to about 10 microns. In some or otherembodiments, combining a conductive material such as, for example,carbon nanotubes, graphene, or metal nanoparticles within one or more ofthe active areas may promote rapid attainment of a steady state current.Suitable amounts of conductive particles may range from about 0.1% toabout 50% by weight of the active area, or from about 1% to about 50% byweight, or from about 0.1% to about 10% by weight, or from about 1% toabout 10% by weight. Stabilizers may also be employed to promoteresponse stability.

It is also to be appreciated that the sensitivity (output current) ofthe analyte sensors toward each analyte may be varied by changing thecoverage (area or size) of the active areas, the areal ratio of theactive areas with respect to one another, the identity and thickness ofthe mass transport limiting membrane overcoating the active areas, andany combination thereof. Variation of these parameters may be conductedreadily by one having ordinary skill in the art once granted the benefitof the disclosure herein.

Although the foregoing description is primarily directed to analytesensors configured for detecting two different analytes, it is to beappreciated that the concepts above may be extended for detecting morethan two analytes using a corresponding number of active areas locatedupon a single working electrode. Specifically, analyte sensors employingmore than two active areas and a corresponding number of differentenzymes (and electron transfer agents) therein may be employed to detecta like number of different analytes in further embodiments of thepresent disclosure. Provided that the oxidation-reduction potential ofeach active area is sufficiently separated from that of other activeareas, the signal contribution from each active area may be analyzed ina manner related to that described above to provide the concentration ofeach analyte.

In more particular embodiments, the first active area may comprise aglucose-responsive enzyme, such as glucose oxidase, and the secondactive area may comprise a lactate-responsive enzyme, such as lactateoxidase, in addition to the suitable electron transfer agents andpolymers discussed in more detail above. According to particularembodiments, analyte sensors suitable for detecting glucose and lactatemay comprise a working electrode having a first active area and a secondactive area disposed thereon, and a mass transport limiting membraneovercoating the first and second active areas upon the workingelectrode, in which the second active area comprises a polymer, analbumin, and a lactate-responsive enzyme (e.g., lactate oxidase)covalently bonded to the polymer and the first active area comprises aglucose-responsive enzyme (e.g., glucose oxidase) covalently bonded to apolymer. First and second electron transfer agents differing from oneanother may be present in each active area. In more specificembodiments, the mass transport limiting membrane may comprise at leasta crosslinked polyvinylpyridine homopolymer or copolymer. Thecomposition of the mass transport limiting membrane may be the same ordifferent where the mass transport limiting membrane overcoats eachactive area. In particular embodiments, the mass transport limitingmembrane overcoating the first active area may be single-component(contain a single membrane polymer) and the mass transport limitingmembrane overcoating the second active area may be multi-component(contain two or more different membrane polymers, one of which is apolyvinylpyridine homopolymer or copolymer), either as a bilayer orhomogeneous admixture.

Similarly, it is also to be appreciated that some analyte sensors of thepresent disclosure having two or more active areas located upon a givenworking electrode may comprise two or more analyte-responsive enzymes inat least one of the active areas. According to more specificembodiments, the two or more analyte-responsive enzymes in a givenactive area may interact in concert to generate a signal proportional tothe concentration of a single analyte. Thus, analyte-responsive enzymesneed not necessarily be present in a 1:1 ratio with a given selection ofanalytes. Analyte sensors containing in concert interacting enzymes aredescribed in further detail hereinbelow.

Accordingly, multi-analyte detection methods employing analyte sensorsfeaturing multiple enzymes arranged upon a single working electrode arealso described herein. In various embodiments, such methods maycomprise: exposing an analyte sensor to a fluid comprising at least oneanalyte. The analyte sensor comprises a sensor tail comprising at leasta working electrode, particularly a single working electrode, and atleast two active areas disposed upon a surface of the working electrode.Each active area comprises an analyte-responsive enzyme and a polymer,and the analyte-responsive enzyme in each active area is different. Eachactive area has an oxidation-reduction potential, and theoxidation-reduction potential of a first active area is sufficientlyseparated from the oxidation-reduction potential of a second active areato allow production of a signal from the first active area independentof production of a signal from the second active area. The methodsadditionally comprise: obtaining a first signal at or above theoxidation-reduction potential of the first active area, such that thefirst signal is proportional to a concentration of a first analyte;obtaining a second signal at or above the oxidation-reduction potentialof the second active area, such that the second signal is a compositesignal comprising a signal contribution from the first active area and asignal contribution from the second active area; and subtracting thefirst signal from the second signal to obtain a difference signal, thedifference signal being proportional to a concentration of the secondanalyte.

In more specific embodiments, the oxidation-reduction potentialassociated with the first active area may be separated from theoxidation-reduction potential of the second active area by at leastabout 100 mV, or by at least about 150 mV, or by at least about 200 mVin order to provide sufficient separation for independent production ofa signal from the first active area.

In some or other more specific embodiments, the fluid is a biologicalfluid and the analyte sensor is exposed to the biological fluid in vivowithin an individual. Suitable biological fluids for analysis withanalyte sensors having at least two different active areas located upona given working electrode may include any of the biological fluidsdiscussed in more detail above.

In some embodiments, the signals associated with each active area may becorrelated to a corresponding analyte concentration by consulting alookup table or calibration curve for each analyte. A lookup table foreach analyte may be populated by assaying multiple samples having knownanalyte concentrations and recording the sensor response at eachconcentration for each analyte. Similarly, a calibration curve for eachanalyte may be determined by plotting the analyte sensor response foreach analyte as a function of the concentration. According to someembodiments, the calibration curve for analyte sensors of the presentdisclosure may be linear.

A processor may determine which sensor response value in a lookup tableis closest to that measured for a sample having an unknown analyteconcentration and then report the analyte concentration accordingly. Insome or other embodiments, if the sensor response value for a samplehaving an unknown analyte concentration is between the recorded valuesin the lookup table, the processor may interpolate between two lookuptable values to estimate the analyte concentration. Interpolation mayassume a linear concentration variation between the two values reportedin the lookup table. Interpolation may be employed when the sensorresponse differs a sufficient amount from a given value in the lookuptable, such as variation of about 10% or greater.

Likewise, according to some or other various embodiments, a processormay input the sensor response value for a sample having an unknownanalyte concentration into a corresponding calibration curve. The sensormay then report the analyte concentration accordingly.

Embodiments of analyte sensors having two different active areasdisposed upon a given working electrode may employ sensor configurationsrelated to those depicted in FIGS. 2A-2C and described above. It is tobe appreciated, however, that suitable analyte sensors may also featuremultiple working electrodes, such as the sensor configuration shown inFIG. 3, with at least one of the working electrodes having at least twoactive areas that differ from one another. It is also to be appreciatedthat other analyte sensor configurations having two or more differentactive areas disposed upon the surface of a given working electrode alsoreside within the scope of the present disclosure. For example, thelocation, orientation or functionality of the working electrode and thecounter and/or reference electrodes may differ from that shown in thefigures herein.

FIG. 4 shows an illustrative analyte sensor configuration compatible foruse in some embodiments of the disclosure herein, in which two differentactive areas are disposed upon the surface of a single workingelectrode. The analyte sensor configuration of FIG. 4 bears mostsimilarity to that of FIG. 2C and may be better understood by referencethereto. Where appropriate, common reference characters from FIG. 2C areused in FIG. 4 in the interest of clarity, and features having a commonstructure and/or function are not described again in further detail inthe interest of brevity. Again, it is to be appreciated that otheranalyte sensor configurations may similarly incorporate the featuresdescribed below for FIG. 4.

Referring to FIG. 4, analyte sensor 400 includes active areas 218 a and218 b upon the surface of working electrode 214. Active area 218 aincludes a first electron transfer agent and a first analyte-responsiveenzyme that may be covalently bonded to a polymer comprising active area218 a. Active area 218 b similarly includes a second electron transferagent and a second analyte-responsive enzyme that may be covalentlybonded to a polymer comprising active area 218 b. The first electrontransfer agent and the second electron transfer agent may differ incomposition so as to provide separation of the oxidation-reductionpotentials of first active area 218 a and second active area 218 b. Inparticular embodiments, active area 218 b may comprise alactate-responsive enzyme, such as lactate oxidase, and active area 218a may comprise a glucose-responsive enzyme, such as glucose oxidase.

The oxidation-reduction potentials of first active area 218 a and secondactive area 218 b may be sufficiently separated from one another toallow production of a signal from first active area 218 a independent ofsignal production from second active area 218 b. As such, analyte sensor400 may be operated at a first potential at which an oxidation-reductionreaction occurs within first active area 218 a but not within secondactive area 218 b. Thus, a first analyte (e.g., glucose) may beselectively detected at or above the oxidation-reduction potential offirst active area 218 a, provided that the applied potential is not highenough to promote an oxidation-reduction reaction with second activearea 218 b. A concentration of the first analyte may be determined fromthe sensor response of first active area 218 a by referring to a lookuptable or calibration curve.

At or above the oxidation-reduction potential of second active area 218b, separate oxidation-reduction reactions may take place simultaneouslyor near simultaneously within both first active area 218 a and secondactive area 218 b. As a result, the signal produced at or above theoxidation-reduction potential of second active area 218 b may comprise acomposite signal having signal contributions from both first active area218 a and second active area 218 b. To determine the concentration ofthe second analyte (e.g., lactate) from the composite signal, the signalfrom first active area 218 a at or above its correspondingoxidation-reduction potential may be subtracted from the compositesignal to provide a difference signal associated with second active area218 b alone. Once the difference signal has been determined, theconcentration of a second analyte may be determined by reference to alookup table or calibration curve.

As mentioned previously, similar considerations also apply to separatinga first signal and a second signal from a single active area containingtwo different analyte-responsive enzymes in order to determine theconcentrations of first and second analytes that differ from oneanother.

In some or other embodiments of the present disclosure, multiple enzymesmay be present in a single active area. Unlike sensor configurations inwhich multiple enzymes function independently to detect differentanalytes, particularly spaced apart in separate active areas upon thesurface of a working electrode, multiple enzymes arranged in a singleactive area may function in concert to facilitate detection of a singleanalyte, according to some embodiments of the present disclosure,particularly in the presence of a stabilizer. As used herein, the term“in concert” and grammatical variants thereof refer to a coupled enzymereaction, in which the product of a first enzymatic reaction becomes thesubstrate for a second enzymatic reaction, and the second enzymaticreaction serves as the basis for measuring the concentration of thesubstrate (analyte) reacted during the first enzymatic reaction. It maybe desirable to utilize two enzymes acting in concert with one anotherto detect a given analyte of interest when a single enzyme is unable tofacilitate detection. Situations in which a single enzyme may beineffective for promoting analyte detection include, for example, thosein which the enzyme is inhibited by one or more products of theenzymatic reaction or is unable to cycle between an oxidized state andreduced state when disposed within an analyte sensor.

As also disclosed herein, multiple enzymes disposed in separate activeareas may likewise interact in concert to promote detection of a singleanalyte. When the multiple enzymes are located in separate active areas,one of the active areas may be isolated from the working electrode sothat electron transfer to and from the working electrode takes placefrom only one of the active areas.

In more specific embodiments, analyte sensors featuring at least twoenzymes interacting in concert with one another may comprise: a sensortail comprising at least a working electrode; and at least one activearea disposed upon a surface of the working electrode. The at least oneactive area comprises a first enzyme, a second enzyme, and a polymer.The first and second enzymes are capable of interacting in concert, suchthat the first enzyme is capable of converting an analyte into a firstproduct, and the second enzyme is capable of converting the firstproduct into a second product to generate a signal at the workingelectrode. The second enzyme is unreactive with the analyte. At leastthe second enzyme is covalently bonded to the polymer in the at leastone active area. Analysis of a signal (e.g., the current measured at afixed input voltage) resulting from the reaction of the first productinto the second product may provide a basis for detecting an analyte andmeasuring its concentration, as explained in further detail hereinafter.

More specifically, the active area may comprise an electron transferagent, such as those described above, and only the second enzyme iscapable of exchanging electrons with the electron transfer agent, inwhich case the first enzyme may indirectly convey electrons to thesecond enzyme, as explained hereinafter. Accordingly, more specificembodiments of the present disclosure may feature a first enzyme that isnot covalently bonded to the polymer (so that it is less likely toexchange electrons with the electron transfer agent) and a second enzymethat is covalently bonded to the polymer (to promote exchange ofelectrons with the electron transfer agent). The electron transfer agentmay be covalently bonded to the polymer in the active area in eithercase. Coordinative bonding is also included within the scope of covalentbonding in accordance with the disclosure herein.

A stabilizer may be present in the active area, according to someembodiments. Particularly suitable stabilizers for analyte sensorscontaining in concert interacting enzymes include catalase and albumin,for example.

According to some embodiments, the sensor tail may be configured forinsertion into a tissue of interest. As such, according to someembodiments, analyte sensors containing enzymes capable of interactingin concert with one another in a given active area may be adapted toanalyze the concentration of an analyte in a biological fluid in vivo.The identity of the biological fluid is again not particularly limited.

As above, sensor configurations incorporating two enzymes that arecapable of interacting in concert may include those in which the atleast one active area comprises an electron transfer agent that iscovalently bonded to the polymer. Again, coordinative bonding is alsoincluded within the scope of covalent bonding in accordance with thedisclosure herein. In such embodiments, at least the second enzyme mayalso be covalently bonded to the polymer. In some embodiments, the firstenzyme is not covalently bonded to the polymer. In other embodiments,both the first enzyme and the second enzyme may be covalently bonded tothe polymer in the at least one active area. Covalent bonding of thefirst enzyme to the polymer may be desirable, for example, to lessen thelikelihood of leaching the first enzyme from the at least one activearea.

Analyte sensor configurations suitable for incorporating two enzymesinteracting in concert with one another in one or more active areas upona working electrode may be similar to those shown in FIGS. 2A-2C anddescribed in more detail above. Enzymes capable of interacting inconcert with one another (i.e., concerted enzymes or concerted enzymepairs) may also be incorporated in analyte sensor configurations havingmultiple working electrodes (FIG. 3) or having multiple active areasarranged on a given working electrode (FIG. 4). Any of the analytesensors disclosed herein with a concerted enzyme pair located directlyupon the surface of a working electrode may employ any of the foregoinganalyte sensor configurations. Analyte sensor configurations having twoor more enzymes interacting in concert with one another in multipleactive areas, in which one of the active areas is remote from theworking electrode, are discussed further below in reference to FIGS. 6Aand 6B.

In more specific configurations of analyte sensors containing concertedenzymes both disposed directly on a working electrode, the first enzymemay be alcohol oxidase (AOX) and the second enzyme may be xanthineoxidase (XOX). With this pair of enzymes, the analyte sensor may befunctional to detect an alcohol, particularly ethanol, according to oneor more embodiments. Cooperativity between alcohol oxidase and xanthineoxidase for detecting ethanol and other alcohols with both enzymesdisposed upon a working electrode is explained in further detailhereinafter (see FIG. 5A). In more specific embodiments of the presentdisclosure, the xanthine oxidase may be covalently bonded to the polymerin the active area, and the alcohol oxidase is not covalently bonded tothe polymer. In still more specific embodiments, both xanthine oxidaseand an electron transfer agent may be covalently bonded to the polymer,and the alcohol oxidase is not covalently bonded to the polymer.Catalase may be present as a stabilizer with this pair of enzymes.

Another pair of concerted enzymes that may be suitable for use in thedisclosure herein with both enzymes disposed directly on the surface ofa working electrode is β-hydroxybutyrate dehydrogenase and diaphorase.This concerted enzyme pair may be used for ketone body detection, withβ-hydroxybutyrate being a representative molecule indicative of thepresence of ketones. In sensor configurations containing this pair ofconcerted enzymes, β-hydroxybutyrate dehydrogenase may convertβ-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD⁺)into acetoacetate and reduced nicotinamide adenine dinucleotide (NADH).The NADH may then undergo reduction under diaphorase mediation, with theelectrons transferred during this process providing the basis for ketonedetection at the working electrode. The concerted reaction betweenβ-hydroxybutyrate and diaphorase (mediated by NAD⁺ cofactor) fordetecting ketones is shown in FIG. 5B. Albumin may be present as astabilizer with this pair of concerted enzymes.

Still other alternative enzymatic detection chemistries for ketones areshown in FIGS. 5C and 5D. As shown in FIG. 5C, β-hydroxybutyratedehydrogenase may convert β-hydroxybutyrate and NAD⁺ into acetoacetateand NADH. Instead of electron transfer to the working electrode beingcompleted by diaphorase (see FIG. 5B), the reduced form of NADH oxidase(NADHOx (Red)) undergoes a reaction to form the corresponding oxidizedform (NADHOx (Ox)). NADHOx (Red) may then be reformed by a reaction withmolecular oxygen to produce superoxide, which may undergo subsequentconversion to hydrogen peroxide under superoxide dismutase (SOD)mediation. The SOD may be covalently bonded to the polymer in the activearea, according to various embodiments. The hydrogen peroxide may thenundergo a reaction at the working electrode to provide a signal that maybe correlated to the amount of ketones that are present. FIG. 5D showsanother alternative enzymatic detection chemistry in whichβ-hydroxybutyrate dehydrogenase again may convert β-hydroxybutyrate andNAD into acetoacetate and NADH. The detection cycle in this case iscompleted by oxidation of poly-1,10-phenanthroline-5,6-dione at theworking electrode. Like other sensing chemistries disclosed herein,inclusion of an albumin in the active area may provide a surprisingimprovement in response stability.

Creatine amidohydrolase and sarcosine oxidase are another pair ofconcerted enzymes that may be suitable for use in the disclosure hereinwhen both enzymes are disposed directly upon a working electrode.Creatine amidohydrolase generates sarcosine and urea from creatine. Thesarosine oxidase, in turn, may catalyze the reaction of sarcosine toform glycine, formaldehyde, and hydrogen peroxide. Accordingly,detection of the hydrogen peroxide at the working electrode may serve asthe basis for quantifying creatine and/or sarcosine.

The detection of ethanol and other alcohols using alcohol oxidase andxanthine oxidase via a concerted enzymatic reaction will now bedescribed in further detail. Alcohol oxidase interacts with ethanol toform acetaldehyde and hydrogen peroxide. Other alcohols react to formaldehydes with a corresponding higher or lower carbon count.Advantageously, alcohol oxidase only catalyzes the forward conversion ofethanol into acetaldehyde (as opposed to performing the reactionreversibly, such as is the case for alcohol dehydrogenase), which may befavorable for use of this enzyme in an analyte sensor. Moreover, alcoholoxidase contains a strongly bound flavin co-factor, such that exogenousco-factors need not necessarily be combined with alcohol oxidase torender the enzyme active for promoting alcohol oxidation.

In principle, alcohol oxidase alone could be employed for ethanoldetection in an analyte sensor by assaying either the acetaldehyde orhydrogen peroxide products produced in the enzymatic reaction. There aretwo issues with this approach, however. First, both acetaldehyde andhydrogen peroxide are inhibitory toward alcohol oxidase. Thus, if thesecompounds are not cleared from the sensor environment, the alcoholoxidase becomes inactive for promoting ethanol oxidation, therebyleaving the analyte sensor non-functional for assaying ethanol.Moreover, if acetaldehyde and hydrogen peroxide become sequestered orundergo quenching with other agents, there is no longer a speciesavailable for electrochemical detection. Second, alcohol oxidase doesnot freely exchange electrons with oxidation-reduction mediators, otherthan molecular oxygen. As such, electron transfer agents associated witha polymer in the active area of an analyte sensor, such as the osmiumand other transition metal complexes discussed herein, are ineffectivefor cycling alcohol oxidase from an inactive reduced state into anoxidized state that is reactive with ethanol. Thus, although alcoholoxidase may be optionally covalently bonded to the polymer, there are noparticular benefits to the electron transfer process in doing so. Thatis, covalent bonding of alcohol oxidase to the polymer does not aid inpromoting electron transfer with the electron transfer agent.

The concerted combination of alcohol oxidase and xanthine oxidasedirectly upon a working electrode, particularly together in a givenactive area, may overcome at least some of the foregoing challengesassociated with ethanol detection using an analyte sensor employingalcohol oxidase. Acetaldehyde and other aldehydes may serve as asubstrate for xanthine oxidase, with the acetaldehyde beingenzymatically converted to acetic acid. Thus, xanthine oxidase may clearacetaldehyde from the sensor environment, thereby precludingacetaldehyde-based inactivation of the alcohol oxidase. Catalase may bepresent in the active area to clear hydrogen peroxide (e.g., as acatalase-hydrogen peroxide complex), thereby precluding inactivation ofthe alcohol oxidase with this species. In addition, unlike alcoholoxidase, xanthine oxidase may exchange electrons with osmium and othertransition metal complexes associated with the polymer in the activearea of the analyte sensor. As such, xanthine oxidase may cycle betweenits oxidized and reduced forms, thereby allowing the analyte sensor tomaintain an active sensing state. Detection of ethanol in the foregoinganalyte sensors is therefore based upon the enzymatic reaction ofxanthine oxidase with acetaldehyde, the enzymatic reaction product ofethanol. Moreover, by configuring the enzymes in the analyte sensor inthe foregoing manner, the alcohol oxidase may undergo re-oxidation withmolecular oxygen to maintain its activity.

FIG. 5A shows the concerted enzymatic reaction cycle associated withethanol detection using alcohol oxidase and xanthine oxidase disposeddirectly upon a working electrode, according to various embodiments ofthe present disclosure. Xanthine oxidase is covalently bonded to apolymer in the active area of the analyte sensor, and alcohol oxidase isnon-covalently associated with the polymer in the active area. Inaddition to xanthine oxidase, an osmium complex or other transitionmetal complex capable of exchanging electrons with this enzyme is alsocovalently bonded to the polymer. As shown in FIG. 5A, ethanol reactswith oxidized (active) alcohol oxidase in the presence of a flavinco-factor (FAD-already bonded with the alcohol oxidase), thereby formingreduced alcohol oxidase, acetaldehyde, and hydrogen peroxide. Thereduced alcohol oxidase may be re-oxidized with molecular oxygen asshown to return the alcohol oxidase to its catalytically active oxidizedform.

Referring still to FIG. 5A, the acetaldehyde enzymatically formed fromethanol then undergoes a subsequent reaction with the oxidized form ofxanthine oxidase in the presence of a flavin co-factor that is presentnatively with the enzyme. Acetic acid is formed in this process and thexanthine oxidase is transformed into a reduced state. The reducedxanthine oxidase may then react with the transition metal electrontransfer agent associated with the polymer to transfer electrons to theworking electrode, thereby producing a current and regenerating theoxidized form of xanthine oxidase. Although not shown in FIG. 5A,hydrogen peroxide is separately cleared from the sensor environment bycatalase that is present in the active area.

As can be appreciated from FIG. 5A, the amount of enzymatically formedacetaldehyde is proportional to the amount of ethanol originallypresent. As such, the current produced at the working electrode duringthe xanthine oxidase oxidation of the acetaldehyde may be proportionalto the amount of acetaldehyde present, and, by extension, the amount ofethanol. Correlation of the working electrode current to the ethanolconcentration may take place by referring to a lookup table of currentsat known ethanol concentrations or by utilizing a calibration curve, theconcepts of which are described in more detail hereinabove.

Similarly, the current produced at the working electrode when analyzingfor ketones may be proportional to the amount of β-hydroxybutyrate thatis oxidized to form acetoacetone (FIGS. 5B-5D). Correlation of thecurrent at the working electrode may therefore take place in a mannerrelated to that provided above for ethanol (e.g., using a calibrationcurve or lookup table).

Accordingly, in more specific embodiments, the present disclosureprovides alcohol sensors based upon a concerted enzymatic reaction ofalcohol oxidase and xanthine oxidase. More specifically, the alcoholsensors may comprise a sensor tail comprising at least a workingelectrode, and at least one active area disposed upon a surface of theworking electrode, wherein the at least one active area comprisesalcohol oxidase, xanthine oxidase, catalase, a polymer, and an electrontransfer agent. The electron transfer agent and the xanthine oxidase maybe covalently bonded to the polymer, and the alcohol oxidase is notcovalently bonded to the polymer, according to particular embodiments.The alcohol oxidase and the xanthine oxidase are capable of interactingin concert to generate a signal at the working electrode that isproportional to an alcohol concentration. More specifically, the alcoholoxidase and the xanthine oxidase are both disposed directly upon theworking electrode in order to accomplish the foregoing.

According to more specific embodiments, the catalase in the at least oneactive area of the alcohol sensors is not covalently bonded to thepolymer. The catalase may be present in an amount ranging from about 1%to about 50% by weight of the polymer, more particularly from about 1%to about 10% by weight of the polymer, or from about 1% to about 5% byweight of the polymer.

As such, the present disclosure also provides detection methods basedupon a concerted enzymatic reaction, in which a concerted enzyme pair isdisposed directly upon the surface of a working electrode. According tovarious embodiments, the detection methods may comprise: exposing ananalyte sensor to a fluid comprising an analyte, the analyte sensorcomprising a sensor tail comprising at least a working electrode and atleast one active area disposed upon a surface of the working electrode,the at least one active area comprising a first enzyme, a second enzyme,and a polymer. The first enzyme and the second enzyme are capable ofinteracting in concert, with the second enzyme being covalently bondedto the polymer and unreactive with the analyte. The methods furtherinclude: reacting the analyte with the first enzyme to form a firstproduct; reacting the first product with the second enzyme to form asecond product to generate a signal at the working electrode; andcorrelating the signal to a concentration of the analyte in the fluid.

According to more specific embodiments, an electron transfer agent mayalso be covalently bonded to the polymer when performing the foregoingmethods. Suitable electron transfer agents are described in more detailabove. In some or other embodiments, the first enzyme is not covalentlybonded to the polymer in the at least one active area, particularly whena covalently bonded electron transfer agent is present.

In more specific embodiments, ethanol detection methods of the presentdisclosure may comprise: exposing an analyte sensor to a fluid,particularly a biological fluid, comprising ethanol, the analyte sensorcomprising a sensor tail comprising at least a working electrode and atleast one active area disposed upon a surface of the working electrodeand comprising alcohol oxidase, xanthine oxidase, catalase, a polymer,and an electron transfer agent. The electron transfer agent and thexanthine oxidase are covalently bonded to the polymer, and the alcoholoxidase is not covalently bonded to the polymer. The alcohol oxidase andthe xanthine oxidase are capable of interacting in concert. The methodsfurther comprise: reacting the ethanol with the alcohol oxidase to formacetaldehyde; reacting the acetaldehyde with the xanthine oxidase toform acetic acid to generate a signal at the working electrode; andcorrelating the signal to a concentration of the ethanol in the fluid.According to some embodiments, the fluid may be a biological fluid andthe analyte sensor may be exposed to the biological fluid in vivo.

Although two different enzymes in a single active area of an analytesensor may interact in concert with one another to determine an analyteconcentration, it is to be appreciated that the enzymes may alsofunction independently of one another to detect alternative analytes inother embodiments. For example, in the case of xanthine oxidase as thesecond enzyme in the analyte sensors described above, instead of usingthe analyte sensor to detect ethanol, the analyte sensor mayalternatively be used to detect any of the wide array of substratescompatible with xanthine oxidase. Alternative substrates for xanthineoxidase may include, for example, hypoxanthine, xanthine, uric acid,purines, pterins, and similar compounds. When the analyte sensors areused in this manner, the alcohol oxidase may remain unused if no alcoholis present, and/or become inactivated by acetaldehyde/hydrogen peroxide,if these species are not cleared by the xanthine oxidase or anotherspecies. Thus, sensors containing concerted enzymes may also beconsidered capable of detecting multiple analytes, one analyte from theconcerted enzyme pair and at least a second analyte from one of themembers of the concerted enzyme pair acting independently. Whether suchsensors assay a single analyte or multiple analytes may be determinedbased upon the environment to which the sensor is exposed.

As referenced above, multiple enzymes disposed in separate active areasmay also interact in concert to promote detection of a single analyte.In some instances, the multiple enzymes may both be located directlyupon the surface of a working electrode, as discussed in more detailabove. In alternative analyte sensor configurations containing multipleenzymes in separate active areas, one of the active areas may beisolated from the working electrode so that electron transfer to theworking electrode takes place from only one of the active areas. Namely,as discussed in further detail hereinbelow, the active area isolatedfrom the working electrode may promote an enzymatic reaction of ananalyte of interest to produce a reaction product (substrate) that isitself reactive with the enzyme in an active area in direct contact withthe working electrode. A signal associated with the enzymatic reactiontaking place in the active area in direct contact with the workingelectrode then provides a basis for detecting the analyte. Correlationof the signal to the analyte concentration may be accomplished in amanner similar to that discussed in more detail above.

More specifically, FIG. 5E shows the concerted enzymatic reaction cycleassociated with ethanol detection using glucose oxidase and xanthineoxidase, as further mediated by catalase, when only the xanthine oxidaseor xanthine oxidase and catalase is disposed upon the surface of aworking electrode, according to various embodiments of the presentdisclosure. The concerted enzymatic reaction cycle shown in FIG. 5E isdependent upon glucose and ethanol being co-present with one another ina fluid during analysis, as explained hereinafter. Since glucose is aubiquitous biological nutrient, it is frequently found co-present withother analytes when assaying a biological fluid. Should a particularfluid undergoing analysis be deficient in glucose, however, certainembodiments of the present disclosure may feature adding glucose to thefluid to promote detection of ethanol or another alcohol using theconcerted enzymatic reaction of glucose oxidase and xanthine oxidase.

Before further discussing the concerted enzymatic reaction depicted inFIG. 5E, illustrative analyte sensor configurations featuring at leastone active area isolated from a working electrode will first bedescribed in further detail. As mentioned previously, the analyte sensorconfigurations shown in FIGS. 2A-4 all feature one or more workingelectrodes having one or more active areas disposed directly upon asurface of each working electrode. In contrast, FIGS. 6A, 6B and 6C showdiagrams of a working electrode in which a first active area is disposeddirectly upon a surface of the working electrode and a second activearea is separated from (spaced apart from or remote from) the workingelectrode by a membrane. The working electrode configurations depictedin FIGS. 6A, 6B and 6C may be substituted for any of the particularworking electrode configurations depicted in FIGS. 2A-4. That is, theworking electrode configurations depicted in FIGS. 6A, 6B and 6C may becombined in any suitable way with a counter electrode and/or a referenceelectrode, membrane, substrates, and similar structures in an analytesensor.

As shown in FIG. 6A, working electrode 400 has active area 402 disposeddirectly upon a surface thereof. Active area 402 comprises a firstenzyme covalently bound to a first polymer. Typically, an electrontransfer agent is also present in active area 402, with the electrontransfer agent also being covalently bound to the polymer. Active area402 is overcoated with membrane 404. Membrane 404 may also overcoat thesurface of working electrode 400, as depicted, as well as other portionsof an analyte sensor in which working electrode 400 is present. Membrane404 isolates active area 406 from working electrode 400, such thatelectron exchange between the two is precluded. Active area 406comprises a second enzyme covalently bound to a second polymer, butwithout a separate electron transfer agent being present. Although FIG.6A shows active area 406 disposed directly over active area 402, it isto be appreciated that they may be laterally spaced apart from oneanother in alternative configurations also compatible with the presentdisclosure. Membrane 408 overcoats active area 406, and optionally othersensor components, to provide mass transport limiting properties.Similarly, as shown in FIG. 6B, membrane 404 need not necessarily extendthe same lateral distance as does membrane 408 upon working electrode400. Indeed, membrane 404 in FIG. 6B overcoats active area 402 but onlya portion of the surface of working electrode 400, with membrane 408overcoating active area 406, the surface of membrane 404 and theremainder of the surface of working electrode 400 not overcoated bymembrane 404. Active areas 402 and 406 may also be laterally offset fromone another in some embodiments, as shown in FIG. 6C, wherein activearea 406 is again isolated from working electrode 400 by membrane 404.

Membrane 408 is permeable to an analyte and any additional componentsneeded to promote an enzymatic reaction in active area 406. Membrane404, in contrast, is permeable to a product formed in active area 406.That is, an analyte reacts in active area 406 to form a first product,which then diffuses through membrane 404 and is subsequently reactedfurther in active area 402 to form a second product or products. Thesecond product is subsequently detectable based on electron exchangewith working electrode 400.

Optionally, lead 410 may extend between active areas 402 and 406 ifglucose detection is desired.

In an alcohol sensor featuring detection based upon a concertedenzymatic reaction of glucose oxidase and xanthine oxidase, the glucoseoxidase is present in active area 406 and the xanthine oxidase ispresent in active area 402. Referring again to FIG. 5E, with continuedreference to FIGS. 6A, 6B and 6C, glucose oxidase is present in activearea 406 and converts exogenous glucose into D-gluconolactone-1,5-dioneand hydrogen peroxide. Unlike alcohol sensors featuring detection basedupon a concerted enzymatic reaction between alcohol oxidase and xanthineoxide (FIG. 5A), the catalase plays a more active role in the concertedenzymatic reaction depicted in FIG. 5E. Namely, catalase reacts with thehydrogen peroxide to form a catalase-hydrogen peroxide complex (the sameperoxide-clearing function exhibited by catalase in the concertedenzymatic reaction of alcohol oxidase and xanthine oxidase), with thecomplex subsequently reacting with ethanol to form acetaldehyde. Theacetaldehyde formed in active area 406 upon reacting ethanol withcatalase-hydrogen peroxide complex diffuses through membrane 404, whichseparates active area 406 from active area 402. Alternately, thecatalase may be present in active area 402, in which case the hydrogenperoxide formed in active area 406 may diffuse through membrane 404 intoactive area 402, form the catalase-hydrogen peroxide complex in activearea 402, and oxidize ethanol to acetaldehyde in active area 402. Onceacetaldehyde has been formed in active area 402, the concerted enzymaticreaction may continue as depicted in FIG. 5E. Membrane 404 may comprisecrosslinked polyvinylpyridine, which is permeable to acetaldehyde. Theacetaldehyde then reacts with xanthine oxidase in active area 402 toform acetic acid in a manner similar to that described above for FIG.5A.

Accordingly, alcohol sensors of the present disclosure may comprise: asensor tail comprising at least a working electrode; a first active areadisposed upon a surface of the working electrode, the first active areacomprising xanthine oxidase, catalase, a first polymer, and an electrontransfer agent; wherein the xanthine oxidase and the electron transferagent are covalently bonded to the first polymer; a first membraneovercoating the first active area, the first membrane comprising a firstmembrane polymer and being permeable to acetaldehyde; a second activearea disposed upon the first membrane, the second active area comprisingglucose oxidase, catalase, and a second polymer; wherein the glucoseoxidase is covalently bonded to the second polymer; and a secondmembrane overcoating the second active area, the second membranecomprising a second membrane polymer and being permeable to glucose andalcohol; wherein the glucose oxidase and the xanthine oxidase arecapable of interacting in concert to generate a signal at the workingelectrode proportional to an alcohol concentration. The alcohol may beethanol in more specific embodiments.

The first membrane polymer and the second membrane polymer may differfrom one another, according to some embodiments. The first membranepolymer may be crosslinked polyvinylpyridine, according to someembodiments. Crosslinked polyvinylpyridine is readily permeable toacetaldehyde in the embodiments of the present disclosure. The secondmembrane polymer may be a crosslinked polyvinylpyridine-co-styrenepolymer, in which a portion of the pyridine nitrogen atoms werefunctionalized with a non-crosslinked poly(ethylene glycol) tail and aportion of the pyridine nitrogen atoms were functionalized with analkylsulfonic acid group. Such second membrane polymers are readilypermeable to both glucose and ethanol.

According to some embodiments, the catalase is not covalently bonded tothe first polymer or the second polymer in the first active area or thesecond active area. The catalase may be physically constrained withinthe first active area and the second active area by any of the firstpolymer, the second polymer, the first membrane polymer, or the secondmembrane polymer.

Similarly, methods for analyzing for ethanol or another alcohol usingthe foregoing analyte sensors comprising in concert interacting glucoseoxidase and xanthine oxidase may comprise: exposing an analyte sensor toa fluid comprising ethanol and glucose; oxidizing the glucose with theglucose oxidase to generate hydrogen peroxide; forming acatalase-hydrogen peroxide complex; oxidizing the alcohol with thecatalase-hydrogen peroxide complex to form acetaldehyde; reacting theacetaldehyde with the xanthine oxidase to form acetic acid to generate asignal at the working electrode; and correlating the signal to aconcentration of the alcohol in the fluid. The fluid may comprise abiological fluid, according to various embodiments of the presentdisclosure. Correlation of the signal to the concentration of alcohol inthe fluid may take place using any suitable correlation techniqueoutlined in more detail above.

In still other embodiments of the present disclosure, multiple enzymesmay be arranged within the active areas of separate working electrodes.As such, the signals associated with the enzymatic reaction occurringwithin each active area may be measured separately by interrogating eachworking electrode at the same time or at different times. The signalassociated with each active area may then be correlated to theconcentration of separate analytes.

As discussed above, a membrane (i.e., a mass transport limitingmembrane) may overcoat one or more of the active areas in an analytesensor in order to increase biocompatibility and to alter the analyteflux to the active areas. Such membranes may be present in any of theanalyte sensors disclosed herein. Because different analytes may exhibitvarying permeability values within a given membrane, an analyte sensorconfigured to analyze for multiple analytes may exhibit dissimilarsensitivities for each analyte. One approach for addressing differingsensitivity values may involve utilizing different membrane thicknessesover each active area. Although feasible, this approach may be difficultto put into practice from a manufacturing standpoint. Namely, it can bedifficult to vary the membrane thickness at different locations usingtypical dip coating techniques that are used for membrane deposition.Another possible approach is to use active areas with different sizesfor each analyte.

Analyte sensors having active areas configured for assaying differentanalytes upon separate working electrodes may address the foregoingissue associated with dissimilar analyte sensitivity. Namely, thedisclosure hereinafter provides various ways in which the membranepermeability may be altered upon each working electrode to levelize theanalyte membrane permeability at each location. That is, the disclosureherein allows the analyte permeability and sensitivity at each workingelectrode to be independently varied. According to the disclosureherein, mass transport limiting membranes comprising two or moredifferent membrane polymers may afford more levelized analytepermeability at each working electrode. Particular membraneconfigurations that may be suitable for levelizing the analytepermeability upon one or more of the working electrodes include bilayermembranes and mixed membranes, each comprising two or more differentmembrane polymers. Surprisingly, bilayer membranes and mixed membranescomprising a membrane polymer that is individually unsuitable forpromoting permeability of a given analyte may provide satisfactoryperformance when located in a bilayer membrane or mixed membrane, asdiscussed hereinafter. This approach may be advantageous compared tovarying the size of the active areas upon each working electrode toprovide comparable sensitivity values for each analyte.

Accordingly, in some embodiments, analyte sensors featuring two or moreenzymes arranged upon separate working electrodes may comprise: a sensortail comprising at least a first working electrode and a second workingelectrode, a first active area located upon a surface of the firstworking electrode, a second active area located upon a surface of thesecond working electrode, a multi-component membrane overcoating thefirst active area, and a homogenous membrane overcoating the secondactive area. The first active area comprises a first polymer and a firstanalyte-responsive enzyme that is reactive with a first analyte, and thesecond active area comprises a second polymer and a secondanalyte-responsive enzyme that is reactive with a second analyte. Thefirst analyte-responsive enzyme and the second analyte-responsive enzymeare different and are reactive with different analytes. Themulti-component membrane comprises at least a first membrane polymer anda second membrane polymer that differ from one another. The homogeneousmembrane comprises one of the first membrane polymer and the secondmembrane polymer.

Particular configurations of the multi-component membranes describedabove may comprise a bilayer membrane in some embodiments or anadmixture of the membrane polymers in other embodiments. Surprisingly,bilayer membranes and admixed membranes may function to levelize theanalyte permeability, as explained in further detail below.

Analyte sensors of the present disclosure having two different activeareas located upon separate working electrodes may employ a sensorconfiguration similar to that described above in FIG. 3 or a variantthereof. For example, in some embodiments, a counter/reference electrodemay replace separate counter and reference electrodes in an analytesensor bearing two or more working electrodes. Similarly, the layerconfiguration and arrangement within analyte sensors having twodifferent active areas located upon separate working electrodes maydiffer from that depicted in FIG. 3. Further details concerning themembrane disposition upon each active area is provided below inreference to FIG. 7.

According to more specific embodiments of the present disclosure,analyte sensors having multiple working electrodes may comprise activeareas in which an electron transfer agent is covalently bonded to thepolymer in each active area. In some or other embodiments, such analytesensors may feature the first analyte-responsive enzyme covalentlybonded to the polymer in the first active area and the secondanalyte-responsive covalently bonded to the polymer in the second activearea. Again, in particular embodiments, the first analyte-responsiveenzyme may be a glucose-responsive enzyme, such as glucose oxidase, andthe second analyte-responsive enzyme may be a lactate-responsive enzyme,such as lactate oxidase.

In still other more specific embodiments, analyte sensors havingmultiple working electrodes may comprise a sensor tail configured forinsertion into a tissue.

In some embodiments, a bilayer membrane may overcoat the first activearea upon one of the working electrodes. The bilayer membrane comprisesa first membrane polymer and a second membrane polymer that are layeredupon one another over the active area. In more specific embodiments, thefirst membrane polymer may be disposed directly upon the active area ofa first working electrode, and the second membrane polymer may bedisposed upon the first membrane polymer to define the bilayer membrane.In such embodiments, the second membrane polymer is present in thehomogenous membrane located upon the second working electrode. Suchbilayer configurations may be prepared, in some embodiments, by coatingthe first membrane polymer only upon the first working electrode (e.g.,by spray coating, painting, inkjet printing, roller coating, or thelike) and then coating the second membrane polymer upon both workingelectrodes at the same time (e.g., by dip coating or a similartechnique). In other embodiments, the bilayer membrane may be configuredas above, with the first membrane polymer being located upon the secondworking electrode.

FIG. 7 shows an illustrative schematic of a portion of an analyte sensorhaving two working electrodes and featuring a bilayer membraneovercoating one of the two working electrodes, which is compatible foruse in some embodiments of the disclosure herein. As shown in FIG. 7,the analyte sensor features sensor tail 600 having working electrodes614 a and 614 b disposed on opposite faces of substrate 612. Active area618 a is disposed upon working electrode 614 a, and active area 618 b isdisposed upon working electrode 614 b. Active areas 618 a and 618 bcontain different analyte-responsive enzymes and are configured to assayfor different analytes, in accordance with the disclosure herein.Although FIG. 7 has shown active areas 618 a and 618 b to be disposedgenerally opposite on another with respect to substrate 612, it is to beappreciate that active areas 618 a and 618 b may be laterally spacedapart (offset) from one another upon opposite faces of substrate 612.Laterally spaced-apart configurations for active areas 618 a and 618 bmay be particularly advantageous for overcoating each active areas 618 aand 618 b with mass transport limiting membranes, as discussedhereinafter.

As further shown in FIG. 7, active area 618 a is overcoated withmembrane layer 620. Membrane layer 620 is a homogenous membranecomprising a single membrane polymer. Active area 618 b is overcoatedwith bilayer membrane 621, which comprises membrane layer 621 a indirect contact with active area 618 b and membrane layer 621 boverlaying membrane layer 621 a. Membrane layers 621 a and 621 bcomprise different membrane polymers. As described above, in particularembodiments, membrane layer 620 and membrane layer 621 b may comprisethe same membrane polymer.

Analyte sensors having multiple active areas upon separate workingelectrodes, in which one of the active areas is overcoated with abilayer membrane, may display levelized or independently variableanalyte permeability, according to one or more embodiments. That is, theanalyte sensors may have sensitivities for two different analytes thatare closer to one another than if the bilayer membrane were not present.In such analyte sensor configurations, the active area overcoated withthe homogeneous membrane (e.g., membrane layer 620 in FIG. 7), mayexhibit analyte permeability for a first analyte that is characteristicof its particular membrane polymer. Surprisingly, a bilayer membrane(e.g., bilayer membrane 621 in FIG. 7) may contain a membrane polymerthat does not negatively impact the permeability of a second analyte(i.e., a membrane polymer having neutral permeability influence),thereby allowing the other membrane polymer comprising the bilayermembrane to exhibit its characteristic permeability for the secondanalyte as if the first membrane polymer was not present. Thus,according to various embodiments, the membrane polymer having neutralpermeability influence and the membrane polymer comprising thehomogeneous membrane may constitute the same polymer.

In some or other specific embodiments, the membrane polymer havingneutral permeability influence may comprise the inner layer of thebilayer membrane. Thus, according to such embodiments, the inner layerof the bilayer membrane and the homogeneous membrane may constitute thesame membrane polymer. In other specific embodiments, the outer layer ofthe bilayer membrane and the homogeneous membrane may constitute thesame membrane polymer.

In particular embodiments, the first active area may comprise aglucose-responsive enzyme, such as glucose oxidase, and the secondactive area may comprise a lactate-responsive enzyme, such as lactateoxidase. Thus, according to such embodiments, the first active areacontaining the glucose-responsive enzyme may be overcoated with thebilayer membrane, and the second active area containing thelactate-responsive enzyme may be overacted with the homogeneous(single-component membrane polymer) membrane. In still more specificembodiments, the second active area may comprise a polymer, an albumin,and a lactate-responsive enzyme covalently bonded to the polymer. In yetstill more specific embodiments, the homogenous membrane overcoating thesecond active area may comprise at least a crosslinked polyvinylpyridinehomopolymer or copolymer, and the bilayer membrane overcoating the firstactive area may also comprise the polyvinylpyridine homopolymer orcopolymer.

In other embodiments of the present disclosure, the multi-componentmembrane may comprise an admixture (homogeneous blend) of the firstmembrane polymer and the second membrane polymer. Such analyte sensorconfigurations may be similar in appearance to that shown in FIG. 7,except for replacement of bilayer membrane 621 with an admixed membranecomprising the two different membrane polymers in a homogeneous blend.As with analyte sensors comprising a bilayer membrane disposed upon oneof the active areas, a homogeneous membrane comprising one of the firstmembrane polymer or the second membrane polymer of the admixed membranemay overcoat the other active area upon the second working electrode.

Similar to a bilayer membrane, an admixed membrane containing a membranepolymer that neutrally influences the permeability of a second analytemay allow the admixed membrane to exhibit permeability for the secondanalyte that is largely characteristic of the other membrane polymer inthe admixture. Thus, according to various embodiments of the presentdisclosure, the membrane polymer of the homogeneous membrane and one ofthe membrane polymers of the admixed membrane may be chosen such thatthe permeability of the second analyte through the admixed membrane isnot substantially altered by the membrane polymer. In particularembodiments, the first active area may comprise a glucose-responsiveenzyme, such as glucose oxidase, and the second active area may comprisea lactate-responsive enzyme, such as lactate oxidase. Thus, according tosuch embodiments, the first active area containing theglucose-responsive enzyme may be overcoated with the admixed membrane,and the second active area containing the lactate-responsive enzyme maybe overacted with the homogeneous (single-component membrane polymer)membrane. In still more specific embodiments, the second active area maycomprise a polymer, an albumin, and a lactate-responsive enzymecovalently bonded to the polymer. In yet still more specificembodiments, the homogenous membrane overcoating the second active areamay comprise at least a crosslinked polyvinylpyridine homopolymer orcopolymer, and the admixed membrane overcoating the first active areamay also comprise the polyvinylpyridine homopolymer or copolymer.

As referenced above, bilayer membranes and admixed membranes maylevelize analyte permeability in analyte sensors of the presentdisclosure, wherein two or more active areas are spatially separatedfrom one another and can be overcoated with different mass transportlimiting membranes. Specifically, bilayer membranes and admixedmembranes of the present disclosure may levelize analyte permeability inanalyte sensors having separate working electrodes and comprising two ormore active areas with different enzymes, with at least one active areabeing located at each working electrode. Thus, such membranes mayadvantageously allow the sensor sensitivity to be varied independentlyfor each analyte. The membrane thickness and/or the relative proportionof the first membrane polymer to the second membrane polymer representother parameters that may be varied to adjust the characteristicpermeability of the analytes at each working electrode.

Accordingly, methods for using an analyte sensor containing two workingelectrodes may comprise exposing an analyte sensor to a fluid comprisingat least one analyte. The analyte sensor comprises a sensor tailcomprising at least a first working electrode and a second workingelectrode. A first active area is disposed upon a surface of the firstworking electrode, and a second active area is disposed upon a surfaceof the second working electrode. The first active area comprises a firstpolymer and a first analyte-responsive enzyme reactive with a firstanalyte, and the second active area comprises a second polymer and asecond analyte-responsive enzyme reactive with a second analyte. Thefirst analyte-responsive enzyme and the second analyte-responsive enzymeare different. A multi-component membrane overcoats the first activearea, and a homogeneous membrane overcoats the second active area. Themulti-component membrane comprises at least a first membrane polymer anda second membrane polymer that differ from one another, and thehomogeneous membrane comprises one of the first membrane polymer or thesecond membrane polymer. The methods further include obtaining a firstsignal at or above an oxidation-reduction potential of the first activearea, obtaining a second signal at or above the oxidation-reductionpotential of the second active area, and correlating the first signal tothe concentration of the first analyte in the fluid and the secondsignal to the concentration of the second analyte in the fluid. Thefirst signal is proportional to the concentration of the first analytein the fluid, and the second signal is proportional to the concentrationof the second analyte in the fluid.

According to more specific embodiments, the first signal and the secondsignal maybe measured at different times. Thus, in such embodiments, apotential may be alternately applied to the first working electrode andthe second working electrode. In other embodiments, the first signal andthe second signal may be measured simultaneously via a first channel anda second channel, in which case a potential may be applied to bothelectrodes at the same time.

Embodiments disclosed herein include:

A. Analyte sensors containing two active areas with differentanalyte-responsive enzymes. The analyte sensors comprise: a sensor tailcomprising at least a working electrode; and at least two active areasdisposed upon a surface of the working electrode, each active areacomprising an analyte-responsive enzyme and a polymer, wherein theanalyte-responsive enzyme in each active area is different; and whereineach active area has an oxidation-reduction potential, and theoxidation-reduction potential of a first active area is sufficientlyseparated from the oxidation-reduction potential of a second active areato allow production of a signal from the first active area independentof production of a signal from the second active area.

B. Methods for assaying two or more analytes using a first active areaand a second active area containing different analyte-responsiveenzymes. The methods comprise: exposing an analyte sensor to a fluidcomprising at least one analyte; wherein the analyte sensor comprises asensor tail comprising at least a working electrode and at least twoactive areas disposed upon a surface of the working electrode, eachactive area comprising an analyte-responsive enzyme and a polymer;wherein the analyte-responsive enzyme in each active area is different;and wherein each active area has an oxidation-reduction potential, andthe oxidation-reduction potential of a first active area is sufficientlyseparated from the oxidation-reduction potential of a second active areato allow production of a signal from the first active area independentof production of a signal from the second active area; obtaining a firstsignal at or above the oxidation-reduction potential of the first activearea, the first signal being proportional to a concentration of a firstanalyte; obtaining a second signal at or above the oxidation-reductionpotential of the second active area, the second signal being a compositesignal comprising a signal contribution from the first active area and asignal contribution from the second active area; and subtracting thefirst signal from the second signal to obtain a difference signal, thedifference signal being proportional to a concentration of the secondanalyte.

C. Analyte sensors containing two or more enzymes that are capable ofinteracting in concert with one another. The analyte sensors comprise: asensor tail comprising at least a working electrode; and at least oneactive area disposed upon a surface of the working electrode, the atleast one active area comprising a first enzyme, a second enzyme, and apolymer, the first enzyme and the second enzyme being capable ofinteracting in concert; wherein the first enzyme is capable ofconverting an analyte into a first product, and the second enzyme iscapable of converting the first product into a second product togenerate a signal at the working electrode; and wherein the secondenzyme is covalently bonded to the polymer and is unreactive with theanalyte.

D. Methods for assaying an analyte using two or more enzymes that arecapable of interacting in concert with one another. The methodscomprise: exposing an analyte sensor to a fluid comprising an analyte;wherein the analyte sensor comprises a sensor tail comprising at least aworking electrode and at least one active area disposed upon a surfaceof the working electrode, the at least one active area comprising afirst enzyme, a second enzyme, and a polymer; wherein the first enzymeand the second enzyme are capable of interacting in concert, and thesecond enzyme is covalently bonded to the polymer and is unreactive withthe analyte; reacting the analyte with the first enzyme to form a firstproduct; reacting the first product with the second enzyme to form asecond product to generate a signal at the working electrode; andcorrelating the signal to a concentration of the analyte in the fluid.

E. Alcohol sensors. The alcohol sensors comprise: a sensor tailcomprising at least a working electrode; and at least one active areadisposed upon a surface of the working electrode, the at least oneactive area comprising alcohol oxidase, xanthine oxidase, catalase, apolymer, and an electron transfer agent; wherein the electron transferagent and the xanthine oxidase are covalently bonded to the polymer, andthe alcohol oxidase is not covalently bonded to the polymer; and whereinthe alcohol oxidase and the xanthine oxidase are capable of interactingin concert to generate a signal at the working electrode proportional toan alcohol concentration.

F. Methods for detecting an alcohol. The methods comprise: exposing ananalyte sensor to a fluid comprising ethanol; wherein the analyte sensorcomprises a sensor tail comprising at least a working electrode and atleast one active area disposed upon a surface of the working electrode,the at least one active area comprising alcohol oxidase, xanthineoxidase, catalase, a polymer, and an electron transfer agent; whereinthe electron transfer agent and the xanthine oxidase are covalentlybonded to the polymer, and the alcohol oxidase is not covalently bondedto the polymer; and wherein the alcohol oxidase and the xanthine oxidaseare capable of interacting in concert; reacting the ethanol with thealcohol oxidase to form acetaldehyde; reacting the acetaldehyde with thexanthine oxidase to form acetic acid to generate a signal at the workingelectrode; and correlating the signal to a concentration of the ethanolin the fluid.

G. Analyte sensors comprising two or more working electrodes that areovercoated with different mass transport limiting membranes. The analytesensors comprise: a sensor tail comprising at least a first workingelectrode and a second working electrode; a first active area disposedupon a surface of the first working electrode, the first active areacomprising a first polymer and a first analyte-responsive enzymereactive with a first analyte; a second active area disposed upon asurface of the second working electrode, the second active areacomprising a second polymer and a second analyte-responsive enzymereactive with a second analyte; wherein the first analyte-responsiveenzyme and the second analyte-responsive enzyme are different; amulti-component membrane overcoating the first active area, themulti-component membrane comprising at least a first membrane polymerand a second membrane polymer that differ from one another; and ahomogeneous membrane overcoating the second active area and differing incomposition from the multi-component membrane, the homogeneous membranecomprising one of the first membrane polymer and the second membranepolymer.

H. Methods for assaying two or more analytes using two workingelectrodes overcoated with different mass transport limiting membranes.The methods comprise: exposing an analyte sensor to a fluid comprisingat least one analyte; wherein the analyte sensor comprises a sensor tailcomprising at least a first working electrode and second workingelectrode; wherein a first active area is disposed upon a surface of thefirst working electrode, the first active area comprising a firstpolymer and a first analyte-responsive enzyme reactive with a firstanalyte, and a second active area is disposed upon a surface of thesecond working electrode, the second active area comprising a secondpolymer and a second analyte-responsive enzyme reactive with a secondanalyte; wherein the first analyte-responsive enzyme and the secondanalyte-responsive enzyme are different; and wherein a multi-componentmembrane overcoats the first active area and a homogeneous membraneovercoats the second active area, the multi-component membranecomprising at least a first membrane polymer and a second membranepolymer that differ from one another, and the homogeneous membranecomprising one of the first membrane polymer or the second membranepolymer and differing in composition from the multi-component membrane;obtaining a first signal at or above an oxidation-reduction potential ofthe first active area, the first signal being proportional to aconcentration of a first analyte in the fluid; obtaining a second signalat or above an oxidation-reduction potential of the second active area,the second signal being proportional to a concentration of the secondanalyte in the fluid; and correlating the first signal to theconcentration of the first analyte in the fluid and the second signal tothe concentration of the second analyte in the fluid.

I. Alcohol sensors comprising glucose oxidase and xanthine oxidaseinteracting in concert. The alcohol sensors comprise: a sensor tailcomprising at least a working electrode; a first active area disposedupon a surface of the working electrode, the first active areacomprising xanthine oxidase, catalase, a first polymer, and an electrontransfer agent; wherein the xanthine oxidase and the electron transferagent are covalently bonded to the first polymer; a first membraneovercoating the first active area, the first membrane comprising a firstmembrane polymer and being permeable to acetaldehyde; a second activearea disposed upon the first membrane, the second active area comprisingglucose oxidase, catalase, and a second polymer; wherein the glucoseoxidase is covalently bonded to the second polymer; and a secondmembrane overcoating the second active area, the second membranecomprising a second membrane polymer and being permeable to glucose andalcohol; wherein the glucose oxidase and the xanthine oxidase arecapable of interacting in concert to generate a signal at the workingelectrode proportional to an alcohol concentration.

J. Methods for detecting an alcohol using a concerted interactionbetween glucose oxidase and xanthine oxidase. The methods comprise:exposing an analyte sensor to a fluid comprising ethanol and glucose;wherein the analyte sensor comprises a sensor tail comprising: at leasta working electrode; a first active area disposed upon a surface of theworking electrode, the first active area comprising xanthine oxidase,catalase, a first polymer, and an electron transfer agent, the xanthineoxidase and the electron transfer agent being covalently bonded to thefirst polymer; a first membrane overcoating the first active area, thefirst membrane comprising a first membrane polymer and being permeableto acetaldehyde; a second active area disposed upon the first membrane,the second active area comprising glucose oxidase, catalase, and asecond polymer, the glucose oxidase being covalently bonded to thesecond polymer; and a second membrane overcoating the second activearea, the second membrane comprising a second membrane polymer and beingpermeable to glucose and alcohol; wherein the glucose oxidase and thexanthine oxidase are capable of interacting in concert; oxidizing theglucose with the glucose oxidase to generate hydrogen peroxide; forminga catalase-hydrogen peroxide complex; oxidizing the alcohol with thecatalase-hydrogen peroxide complex to form acetaldehyde; reacting theacetaldehyde with the xanthine oxidase to form acetic acid to generate asignal at the working electrode; and correlating the signal to aconcentration of the alcohol in the fluid.

Each of embodiments A and B may have one or more of the followingadditional elements in any combination:

Element 1: wherein the sensor tail is configured for insertion into atissue.

Element 2: wherein the oxidation-reduction potential of the first activearea is separated from the oxidation-reduction potential of the secondactive area by at least about 100 mV.

Element 3: wherein the first active area comprises a first electrontransfer agent and the second active area comprises a second electrontransfer agent, the first and second electron transfer agents beingdifferent.

Element 4: wherein the first electron transfer agent is covalentlybonded to the polymer in the first active area and the second electrontransfer agent is covalently bonded to the polymer in the second activearea.

Element 5: wherein the analyte-responsive enzyme in each active area iscovalently bonded to the polymer.

Element 6: wherein the analyte sensor further comprises: a masstransport limiting membrane overcoating at least the at least two activeareas.

Element 7: wherein at least one of the at least two active areascomprises two or more analyte-responsive enzymes, the two or moreanalyte-responsive enzymes interacting in concert to generate a signalproportional to the concentration of a single analyte.

Element 8: wherein the fluid is a biological fluid and the analytesensor is exposed to the biological fluid in vivo.

Element 9: wherein a mass transport limiting membrane overcoats at leastthe at least two active areas.

Element 10: wherein the analyte sensor comprises glucose oxidase as afirst enzyme and lactate oxidase as a second enzyme.

Each of embodiments C and D may have one or more of the followingadditional elements in any combination:

Element 11: wherein the first enzyme is alcohol oxidase and the secondenzyme is xanthine oxidase.

Element 12: wherein the at least one active area further comprisescatalase.

Element 13: wherein the catalase is not covalently bonded to thepolymer.

Element 14: wherein the alcohol oxidase is not covalently bonded to thepolymer.

Element 15: wherein the first enzyme is not covalently bonded to thepolymer.

Element 16: wherein the at least one active area comprises an electrontransfer agent that is covalently bonded to the polymer.

Element 17: wherein the sensor tail is configured for insertion into atissue.

Element 18: wherein the analyte sensor further comprises: a masstransport limiting membrane overcoating at least the at least one activearea.

Element 19: wherein the fluid is a biological fluid and the analytesensor is exposed to the biological fluid in vivo.

Element 20: wherein a mass transport limiting membrane overcoats the atleast one active area.

Each of embodiments E and F may have one or more of the followingadditional elements in any combination:

Element 21: wherein the catalase is not covalently bonded to thepolymer.

Element 22: wherein the sensor tail is configured for insertion into atissue.

Element 23: wherein the alcohol sensor further comprises: a masstransport limiting membrane overcoating at least the at least one activearea.

Element 24: wherein the fluid is a biological fluid and the analytesensor is exposed to the biological fluid in vivo.

Element 25: wherein a mass transport limiting membrane overcoats the atleast one active area.

Each of embodiments G and H may have one or more of the followingadditional elements in any combination:

Element 26: wherein the multi-component membrane comprises a bilayermembrane.

Element 27: wherein the first membrane polymer is disposed directly uponthe first active area, and the second membrane polymer is disposed uponthe first membrane polymer to define the bilayer membrane, the secondmembrane polymer also being present in the homogeneous membrane.

Element 28: wherein the multi-component membrane comprises an admixtureof the first membrane polymer and the second membrane polymer.

Element 29: wherein the sensor tail is configured for insertion into atissue.

Element 30: wherein each active area further comprises an electrontransfer agent that is covalently bonded to the polymer.

Element 31: wherein the first analyte-responsive enzyme is covalentlybonded to the polymer in the first active area, and the secondanalyte-responsive enzyme is covalently bonded to the polymer in thesecond active area.

Element 32: wherein an electron transfer agent is covalently bonded tothe polymer in each active area.

Element 33: wherein the analyte-responsive enzyme in each active area iscovalently bonded to the polymer.

Element 34: wherein the fluid is a biological fluid and the analytesensor is exposed to the biological fluid in vivo.

Element 35: wherein the first signal and the second signal are measuredat different times.

Element 36: wherein the first signal and the second signal are measuredsimultaneously via a first channel and a second channel.

Each of embodiments I and J may have one or more of the followingadditional elements in any combination:

Element 37: wherein the catalase is not covalently bonded to the firstpolymer or the second polymer.

Element 38: wherein the sensor tail is configured for insertion into atissue.

Element 39: wherein the first membrane polymer and the second membranepolymer differ from one another.

Element 40: wherein the first membrane polymer comprises a crosslinkedpolyvinylpyridine.

Element 41: wherein the fluid is a biological fluid and the analytesensor is exposed to the biological fluid in vivo.

By way of non-limiting example, exemplary combinations applicable to A-Jinclude:

The analyte sensor of A in combination with elements 1 and 2; 1 and 3;1, 3 and 4; 1 and 5; 1 and 6; 1 and 7; 2 and 3; 2-4; 2 and 5; 2 and 6; 2and 7; 3-5; 3 and 4; 3 and 5; 3 and 6; 3 and 7; 5 and 6; 6 and 7; 2, 3and 5; 2, 3 and 6; 2-5; and 2, 5 and 6. The method of B in combinationwith elements 2 and 3; 2-4; 2 and 5; 2 and 6; 2 and 7; 2 and 8; 3-5; 3and 4; 3 and 5; 3 and 6; 3 and 7; 3 and 8; 5 and 6; 5 and 8; 6 and 7; 6and 8; 7 and 8; 2, 3 and 5; 2, 3, 5 and 8; 2, 3 and 6; 2, 3, 6 and 8;2-5; 2-5 and 8; 2, 5 and 6; 2, 5, 6 and 8; 2, 3 and 8; 3, 5 and 8; 2, 6and 8; 2, 5 and 8; any one of 2-8 and 9; and any one of 2-8 and 10.

The analyte sensor of C in combination with elements 11 and 12; 11, 12and 13; 12 and 13; 11 and 14; 11 and 15; 11 and 17; 11 and 18; 12 and13; 12 and 14; 12 and 15; 12 and 16; 12 and 17; 12 and 18; 15 and 16; 15and 17; 15 and 18; 16 and 17; 16 and 18; 17 and 18; 11, 12 and 13; 11,12 and 14; 11-14; 11-14 and 16; 11-14 and 17; 11-14 and 18; 15-17;15-18; and 15, 17 and 18. The method of D in combination with elements11 and 12; 11, 12 and 13; 12 and 13; 11 and 14; 11 and 15; 11 and 17; 11and 18; 11 and 19; 11 and 20; 12 and 13; 12 and 14; 12 and 15; 12 and16; 12 and 17; 12 and 18; 12 and 19; 12 and 20; 15 and 16; 15 and 17; 15and 18; 15 and 19; 15 and 20; 16 and 17; 16 and 18; 16 and 19; 16 and20; 17 and 18; 17 and 19; 17 and 20; 18 and 19; 18 and 20; 19 and 20;11, 12 and 13; 11, 12, 13 and 19; 11, 12, 13 and 20; 11, 12 and 14; 11,12, 14 and 19; 11, 12, 14 and 20; 11-14; 11-14 and 19; 11-14 and 20;11-14 and 16; 11-14, 16 and 19; 11-14, 16 and 20; 11-14 and 17; 11-14,17 and 19; 11-14, 17 and 20; 11-14 and 18; 11-14, 18 and 19; 11-14, 18and 20; 15-17; 15-17 and 19; 15-17 and 20; 15-18; 15-18 and 19; 15-18and 20;15, 17 and 18; 15, 17, 18 and 19; 15, 17, 18 and 20; any one of11-16 and 19; and any one of 11-16 and 20.

The analyte sensor of E in combination with elements 21 and 22; 21 and23; 22 and 23; and 21-23. The method of F in combination with elements21 and 22; 21 and 23; 22 and 23; 21-23; 21 and 24; 21 and 25.

The analyte sensor of G in combination with elements 26 and 27; 26 and29; 26, 27 and 29; 26 and 30; 26, 27 and 30; 26 and 31; 26, 27 and 31;28 and 29; 28 and 30; 28-30; 28 and 31; 29 and 30; 29 and 31; and 30 and31. The method of H in combination with elements 26 and 27; 26 and 29;26, 27 and 29; 26 and 30; 26, 27 and 30; 26 and 31; 26, 27 and 31; 28and 29; 28 and 30; 28-30; 28 and 31; 29 and 30; 29 and 31; 30 and 31; 26and 33; 26, 27 and 33; 26 and 34; 26, 27 and 34; 26 and 35; 26, 27 and35; 26 and 36; 26, 27 and 36; 28 and 33; 28 and 34; 28 and 35; 28 and36; 30 and 33; 30 and 34; 30 and 35; 30 and 36; 33 and 34; 33 and 35; 33and 36; 34 and 35; and 34 and 36.

The analyte sensor of I in combination with elements 37 and 38; 37 and39; 37 and 40; 38 and 39; 38 and 40; and 39 and 40. The method of J incombination with elements 37 and 39; 37 and 40; 37 and 41; 39 and 40; 39and 41; and 40 and 41.

Further embodiments disclosed herein include:

A1: Analyte sensors having a multi-component membrane. The analytesensors comprise: a sensor tail configured for insertion into a tissue,the sensor tail comprising at least a working electrode; and first andsecond active areas disposed on the sensor tail, the first and secondactive areas comprising at least two different enzymes for measuring aconcentration of at least one analyte; wherein the first active area isovercoated with a first membrane polymer and a second membrane polymerthat differ from one another.

B1: Analyte sensors having a two active areas on a working electrode andconfigured for sensing different analytes. The analyte sensors comprise:a sensor tail configured for insertion into a tissue and comprising atleast a working electrode; and at least two active areas disposed on thesensor tail, each active area comprising an enzyme, an electron transferagent, and a polymer;

-   -   wherein the enzyme in each active area is different and        responsive to different analytes; and wherein each active area        has an oxidation-reduction potential, and the        oxidation-reduction potential of a first active area is        sufficiently separated from the oxidation-reduction potential of        a second active area to allow production of a signal from the        first active area independent of production of a signal from the        second active area.

Embodiment A1 may have one or more of the following additional elementsin any combination:

Element 1′: wherein the first active area is overcoated with anadmixture of the first membrane polymer and the second membrane polymer,and one of the first membrane polymer and the second membrane polymerovercoats the second active area as a homogenous membrane.

Element 2′: wherein the first active area is overcoated with a bilayermembrane comprising the first membrane polymer disposed upon the secondmembrane polymer, and the second membrane polymer overcoats the secondactive area as a homogenous membrane.

Element 3′: wherein the first active area comprises a first enzyme ofthe at least two different enzymes and the second active area comprisesa second enzyme of the at least two different enzymes.

Element 4′: wherein first enzyme is unreactive with the at least oneanalyte, and the first and second enzymes are capable of interacting inconcert to generate a signal proportional to the concentration of the atleast one analyte.

Element 5′: wherein the second enzyme is capable of converting the atleast one analyte into a product reactive with the first enzyme, suchthat the first enzyme is capable of reacting the product to generate asignal at the working electrode.

Element 6′: wherein the first enzyme is xanthine oxidase and the secondenzyme is glucose oxidase, at least one of the first active area and thesecond active area further comprising catalase.

Element 7′: wherein the catalase is present in the first active area.

Element 8′: wherein the first active area is disposed directly upon theworking electrode and further comprises an electron transfer agent.

Element 9′: wherein the first membrane polymer is disposed directly uponthe first active area, the second active area is disposed directly uponthe first membrane polymer, and the second membrane polymer is disposeddirectly upon the second active area.

Element 10′: wherein the sensor tail comprises a first working electrodeand a second working electrode, the first active area is disposed on asurface of the first working electrode, the second active area isdisposed on a surface of the second working electrode, the first enzymeis reactive with a first analyte to generate a signal proportional tothe concentration the first analyte, and the second enzyme is reactivewith a second analyte to generate a signal proportional to theconcentration the second analyte.

Element 11′: wherein each of the first and second active areas has anoxidation-reduction potential, and the oxidation-reduction potential ofthe first active area is sufficiently separated from theoxidation-reduction potential of the second active area to allowproduction of a signal from the first active area independent ofproduction of a signal from the second active area.

Element 12′: wherein the oxidation-reduction potential of the firstactive area is separated from the oxidation-reduction potential of thesecond active area by at least about 100 mV.

Element 13′: wherein the signal from the first active area correspondsto the first analyte concentration and the signal from the second activearea corresponds to the second analyte concentration.

Element 14′: wherein the first active area comprises a first electrontransfer agent and the second active area comprises a second electrontransfer agent different from the first electron transfer agent.

Embodiment B1 may have one or more of the following additional elementsin any combination:

Element 15′: wherein the oxidation-reduction potential of the firstactive area is separated from the oxidation-reduction potential of thesecond active area by at least about 100 mV.

Element 16′: wherein the first active area comprises a first electrontransfer agent and the second active area comprises a second electrontransfer agent different from the first electron transfer agent.

Element 17′: wherein the first and second active areas are overcoatedwith a mass transport limiting membrane, the first active area beingovercoated with a single membrane polymer and the second active areabeing overcoated with two or more different membrane polymers.

To facilitate a better understanding of the embodiments describedherein, the following examples of various representative embodiments aregiven. In no way should the following examples be read to limit, or todefine, the scope of the invention.

EXAMPLES

Example 1: Detection of Glucose and Lactate Using an Analyte SensorHaving Two Different Active Areas on a Single Working Electrode. Twosolutions containing different poly(vinylpyridine)-bound transitionmetal complexes were prepared. The structure of the polymer of the firstsolution is shown in Formula 1, and the structure of the polymer of thesecond solution is shown in Formula 2. Further details concerning thesepolymers is provided in commonly owned U.S. Pat. No. 6,605,200, whichwas incorporated by reference above. The subscripts for each monomerrepresent illustrative atomic ratios.

The oxidation-reduction potential of the Formula 1 polymer with respectto an Ag/AgCl reference was -50 mV, and the oxidation-reductionpotential of the Formula 2 polymer with respect to the same referencewas +220 mV (270 mV separation, see FIG. 8). In addition to thetransition metal complexes serving as respective electron transferagents, the Formula 1 polymer included glucose oxidase (GOX) covalentlybonded thereto, and the Formula 2 polymer included lactose oxidase (LOX)covalently bonded thereto following deposition on a working electrodeand curing. Crosslinking was accomplished with polyethylene glycoldiglycidyl ether (PEGDE400). Solutions containing the Formula 1 polymerand the Formula 2 polymer were formulated as specified in Tables 1 and 2below.

TABLE 1 Glucose Oxidase (GOX) Formulation in 10 mM HEPES Buffer at pH =8 Initial Final Concentration Added Volume Concentration Component(mg/mL) (mL) (mg/mL) GOX 40 0.41 16.4 Formula 1 Polymer 40 0.34 13.6PEGDE400 40 0.25 10

TABLE 2 Lactate Oxidase (LOX) Formulation in 10 mM MES Buffer at pH =5.5 Initial Final Concentration Added Volume Concentration Component(mg/mL) (μL) (mg/mL) LOX 80 15 20 Albumin 80 10 13 Formula 2 Polymer 4020 13 PEGDE400 40 15 10

To deposit each active area, ˜20 nL of each solution was deposited upona carbon working electrode to form two discrete, separate spots eachhaving an area of approximately 0.1 mm². One spot contained the glucoseoxidase formulation and the other spot contained the lactose oxidaseformulation. Following deposition, the working electrode was curedovernight at 25° C.

After curing, a membrane was deposited upon the working electrode. Themembrane polymer was a polyvinylpyridine copolymer having amine-freepolyether side chain functionalization, as described in U.S. ProvisionalPatent Application 62/684,438, filed on Jun. 13, 2018 and entitled“Temperature-Insensitive Membrane Materials and Analyte SensorsContaining the Same.” Membrane deposition was accomplished by dipcoating the electrode three times in a solution containing 4 mL of themembrane polymer (120 mg/mL) and 0.35 mL PEG1000 (200 mg/mL). Spraycoating, screen printing, or similar processes may be alternately usedto deposit the membrane. Following deposition, the electrode was curedovernight at 25° C. and then further cured in a desiccated vial at 56°C. for two days.

After fabrication, the electrode was analyzed by cyclic voltammetry in abuffer solution containing neither glucose nor lactate. The resultingcyclic voltammogram is shown in FIG. 8, which shows the anodic andcathodic peaks characteristic of the two osmium complexes, since therewas no current contribution from either glucose or lactate. Theoxidation-reduction potentials reported above were calculated from theaverage of the cathodic and anodic peaks for each osmium complex.

To analyze for glucose and lactate, the electrode was placed at apotential above the average oxidation-reduction potential of the firstpolymer, specifically +40 mV (E1 in FIG. 8). At this potential,oxidation of the osmium complex in the first polymer and glucose mayoccur, but not oxidation of the osmium complex in the second polymer orlactate. To oxidize both osmium complexes and both glucose and lactate,the electrode was placed at a potential above the averageoxidation-reduction potential of the second polymer, specifically +250mV (E2 in FIG. 8).

Glucose and lactate analyses were conducted by immersing the electrodein a buffer solution containing 5 mM glucose and 5 mM lactate, and theE1 and E2 potentials were successively applied. FIG. 9 shows fourreplicates of the electrode response in 5 mM glucose/5 mM lactate bufferwhen cycled between E1 and E2. As shown, the current at E1 was about 5nA, which is due to glucose oxidation, and the current at E2 was about10.5 nA, which is due to both glucose and lactate oxidation. Taking thedifference between the measured currents at E1 and E2 provides acontribution of about 5.5 nA at E2 from lactate oxidation. Unknownglucose and lactate concentrations may be analyzed similarly bycomparison to a lookup table or calibration curve.

Example 2A: Detection of Ethanol Using an Analyte Sensor Having TwoDifferent Enzymes Operating In Concert on a Single Working Electrode(XOX/AOX). A spotting solution having the formulation shown in Table 3was prepared. All of the components were dissolved in 10 mM HEPES bufferat pH 8. Crosslinking was accomplished with polyethylene glycoldiglycidyl ether.

TABLE 3 Alcohol Oxidase and Xanthine Oxidase Solution ComponentConcentration (mg/mL) XOX 16 AOX 64 Catalase 4 PVI (pH = 5.8) 16 Oscomplex 10 PEGDE400 10˜15 nL of the solution was deposited on a carbon working electrode as asingle spot having an area of approximately 0.05 mm². Followingdeposition, the working electrode was cured overnight at 25° C.

After curing, a poly(4-vinylpyridine) (PVP) membrane was deposited uponthe working electrode from a coating solution containing 100 mg/mL PVPand 100 mg/mL PEGDE400. Membrane deposition was accomplished by dipcoating the electrode three times in the coating solution. Followingdeposition, the electrode was cured overnight at 25° C. and then furthercured in desiccated vials at 56° C. for two days. Spray coating, screenprinting, or similar processes may be alternately used to deposit themembrane.

Ethanol analyses were conducted by immersing the electrode inethanol-containing PBS solutions each containing varying concentrationsof ethanol. FIG. 10 shows three replicates of the response for anelectrode containing alcohol oxidase and xanthine oxidase together in asensing spot upon exposure to varying ethanol concentrations. As shown,the current response increased over the course of several minutesfollowing exposure to a new ethanol concentration before stabilizingthereafter. FIG. 11A shows an illustrative plot of average currentresponse versus ethanol concentration. FIG. 11B shows corresponding datafor a single sensor. As shown, the sensor response was approximatelylinear over an ethanol concentration range of 0-10 mM.

Example 2B: Detection of Ethanol Using an Analyte Sensor Having TwoDifferent Enzymes Operating In Concert on a Single Working Electrode(XOX/GOX). A first spotting solution having the formulation shown inTable 4 was prepared. All of the components were dissolved in 10 mMHEPES buffer at pH 8. Crosslinking was accomplished with polyethyleneglycol diglycidyl ether.

TABLE 4 Xanthine Oxidase Solution Component Concentration (mg/mL) XOX 25Catalase 12 PVI (pH = 5.8) 12 Os complex 8 PEGDE400 6˜15 nL of the first spotting solution was deposited on a carbon workingelectrode as a single spot (XOX spot) having an area of approximately0.05 mm². Following deposition, the working electrode was curedovernight at 25° C.

After curing, a poly(4-vinylpyridine) (PVP) membrane was deposited uponthe working electrode and the XOX spot from a coating solutioncontaining 100 mg/mL PVP and 100 mg/mL PEGDE400. Membrane deposition wasaccomplished by dip coating the electrode three times in the coatingsolution. Spray coating, screen printing, or similar processes may bealternately used to deposit the membrane. Following deposition, theelectrode was cured overnight at 25° C. and then further cured indesiccated vials at 56° C. for two days.

A second spotting solution having the formulation shown in Table 5 wasprepared. All of the components were dissolved in 10 mM HEPES buffer atpH 8. Crosslinking was accomplished with polyethylene glycol diglycidylether.

TABLE 5 Glucose Oxidase Solution Component Concentration (mg/mL) GOX 16Catalase 32 PVI (pH = 5.8) 32 PEGDE400 6˜15 nL of the second spotting solution was deposited on the PVP membranefrom above as a single spot (GOX spot) having an area of approximately0.05 mm². Following deposition, curing was performed overnight at 25° C.

After curing, a second membrane was deposited upon the GOX spot and thePVP membrane. The membrane polymer in this case was a crosslinkedpolyvinylpyridine-co-styrene polymer, in which a portion of the pyridinenitrogen atoms were functionalized with a non-crosslinked poly(ethyleneglycol) tail and a portion of the pyridine nitrogen atoms werefunctionalized with an alkylsulfonic acid group. The membrane at thislocation was deposited from a coating solution comprising 35 mg/mL ofthe crosslinked polyvinylpyridine-co-styrene polymer and 100 mg/mLPEGDE400. Membrane deposition was accomplished by dip coating theelectrode three times in the coating solution. Spray coating, screenprinting, or similar processes may be alternately used to deposit themembrane. Following deposition, the electrode was cured overnight at 25°C. and then further cured in desiccated vials at 56° C. for two days.

Ethanol analyses were conducted by immersing the electrode inethanol-containing PBS solutions each containing varying concentrationsof ethanol. FIG. 12A shows two replicates of the response for anelectrode containing glucose oxidase and xanthine oxidase layered inseparate active areas and spaced apart by a membrane upon exposure tovarying ethanol concentrations, in which catalase is in the active areawith the glucose oxidase. As shown, the current response increased overthe course of several minutes following exposure to a new ethanolconcentration before stabilizing thereafter. Good reproducibility beenthe two replicates was observed. FIG. 13 shows an illustrative plot ofaverage current response versus ethanol concentration. The curve shapewas similar to that obtained using AOX/XOX (FIG. 11A, Example 2A).

FIG. 12B shows comparative response data between an electrode containingglucose oxidase and xanthine oxidase layered in separate active areasand spaced apart by a membrane upon exposure to varying ethanolconcentrations, in which catalase is present in the active areasseparately. As shown, the sensor response was greater when the catalasewas included in the active area containing xanthine oxidase.

Example 3: Comparative Analyte Sensor Response Toward Lactate in thePresence of Various Mass Transport Limiting Membranes. For this example,the membrane formulations below were coated onto a carbon workingelectrode containing lactate oxidase in an active area thereof. Theactive area was deposited using the lactate oxidase formulation asdescribed in Example 1, except substituting Formula 1 Polymer forFormula 2 Polymer in the formulation and adjusting the concentrations tothose specified in Table 6 below.

TABLE 6 Lactate Oxidase (LOX) in 10 mM MES Buffer at pH = 5.5 ComponentConcentration (mg/mL) LOX 24.6 Albumin 24.6 Formula 1 Polymer 9.2PEGDGE400 6.2Active area deposition and curing was performed as described in Example1, except for depositing six spots each having an area of 0.01 mm²instead of the single spot having an area of 0.1 mm² in Example 1.Unless otherwise indicated below, membrane deposition was performed bydip coating (1-5 dips of the electrode and a wait time of about 10minutes between dips). Following the completion of dip coating, themembranes were cured for 24 hours at 25° C., followed by 48 hours at 56°C. in desiccated vials.

Electrode response was measured by placing the active area of theelectrode in a beaker containing 100 mM pH=7.5 phosphate buffered salineat 37° C. The potential was raised to +40 mV versus Ag/AgCl, and thecurrent was monitored continuously thereafter. To determine the responseat various lactate concentrations, sodium lactate was added to thebuffer solution in increments of 1 mM up to 5 mM. To determine theresponse stability, the current was measured in 5 mM sodium lactate overan extended period, such as two weeks.

Membrane Polymers 1A and 1B: The first tested membrane polymer was acrosslinked polyvinylpyridine-co-styrene polymer, in which a portion ofthe pyridine nitrogen atoms were functionalized with a non-crosslinkedpoly(ethylene glycol) tail and a portion of the pyridine nitrogen atomswere functionalized with an alkylsulfonic acid group. Two differentcrosslinking agents were used to affect crosslinking of this membranepolymer: glycerol triglycidyl ether (Gly3-Formulation 1) andpolyethylene glycol diglycidyl ether 400 (PEGDGE400-Formulation 2).Formulation 1 contained 4 mL of the membrane polymer in 80:20ethanol:HEPES buffer (140 mg/mL), 1 mL of Gly3 in 80:20 ethanol:HEPESbuffer (35 mg/mL), and 0.0132 mL of aminopropyl-terminatedpolydimethylsiloxane (PDMS) in ethanol (100 mg/mL). Formulation 2contained 4 mL of the membrane polymer in 80:20 ethanol:HEPES buffer(140 mg/mL), 0.2 mL of PEGDGE400 in 80:20 ethanol:HEPES buffer (100mg/mL), and 0.0132 mL of aminopropyl-terminated polydimethylsiloxane(PDMS) in ethanol (100 mg/m L). The corresponding crosslinked polymersare designated as Polymers 1A and 1B, respectively, herein.

FIG. 14 shows an illustrative plot of the response of an electrodeovercoated with Polymers 1A and 1B to a 5 mM lactate solution. As shown,neither formulation afforded a stable sensor response over time. Thesensor current afforded by Polymer 1A (Formulation 1) decreased slowlyover a two-week measurement time, whereas the sensor current afforded byPolymer 1B (Formulation 2) initially increased over the first week oflactate exposure and then decreased. In contrast, both of thesemembranes provided a stable response in the presence of glucose analyte(data not shown).

Membrane Polymer 2: The second tested membrane polymer waspolyvinylpyridine (PVP) crosslinked with polyethylene glycol diglycidylether 1000 (PEGDGE1000). This membrane polymer is designated as Polymer2 herein. The membrane formulation (Formulation 3) contained 4.3 mL ofPVP in 80:20 ethanol:HEPES buffer (100 mg/mL), 0.25 mL of PEGDGE1000 in80:20 ethanol:HEPES buffer (200 mg/mL), and 0.0132 mL of PDMS in ethanol(100 mg/mL).

FIG. 15 shows an illustrative plot of the response of an electrodeovercoated with Polymer 2 (Formulation 3) to a 5 mM lactate solution.Like Polymers 1A and 1B, Polymer 2 also did not afford a stable currentresponse over time. There was a large decrease in response over thefirst 48 hours, followed by relatively stable performance thereafter. Inaddition, the sensitivity was well below a target value of about 1nA/mM. Like Polymers 1A and 1B, Polymer 2 provided a stable currentresponse in the presence of glucose analyte (data not shown).

Membrane Polymer 3: The third tested membrane polymer waspolyvinylpyridine (PVP) crosslinked with PEGDGE400. This membranepolymer is designated as Polymer 3 herein. The membrane formulation(Formulation 4) contained 4.3 mL of PVP in 80:20 ethanol:HEPES buffer(100 mg/mL), 0.23 mL of PEGDGE400 in 80:20 ethanol:HEPES buffer (100mg/mL), and 0.0132 mL of PDMS in ethanol (100 mg/mL).

FIG. 16 shows an illustrative plot of the response of an electrodeovercoated with Polymer 3 (Formulation 4) to a 5 mM lactate solution.Unlike Polymer 2, which was crosslinked with a higher molecular weightvariant of the same crosslinking agent, Polymer 3 surprisingly affordeda stable current response over time. Moreover, the current respondedrapidly and achieved a stable current as increasing amounts of lactatewere added in 1 mM increments (FIG. 17).

Membrane Polymer 4: The fourth tested membrane polymer was PVPcontaining 3-4 wt. % non-crosslinked PEG side chains, which was thencrosslinked with PEGDGE1000. Thus, the tested membrane polymer containedboth non-crosslinked PEG chains and crosslinking PEG1000 chains. Thismembrane polymer is designated as Polymer 4 herein. The membraneformulation (Formulation 5) contained 4.3 mL of the polymer in 80:20ethanol:HEPES buffer (100 mg/mL), 0.025 mL of PEGDGE1000 in 80:20ethanol:HEPES buffer (200 mg/mL), and 0.0132 mL of PDMS in ethanol (100mg/mL).

FIG. 18 shows an illustrative plot of the response of an electrodeovercoated with Polymer 4 (Formulation 5) to a 5 mM lactate solution.Unlike Polymers 2 and 3, Polymer 4 surprisingly afforded a stablecurrent response over time. Moreover, the current responded rapidly andachieved a stable value as increasing amounts of lactate were added in 1mM increments (FIG. 19).

Bilayer Membranes Comprising Polymers 2 and 1B or Polymers 2 and 1A:Formulation 3 (Polymer 2) was coated onto the electrode surface byrepeated dip coating operations. Spray coating, screen printing, orsimilar processes may be alternately used to deposit the membrane.Formulation 2 (Polymer 1B) was then coated onto the depositedcrosslinked PVP layer by repeated dip coating operations. There was a 10minute wait time between successive dips. After all dipping operationswere complete, the sensors were cured at 25° C. for 24 hours, followedby 48 hours at 56° C. in desiccated vials. As shown above, neither ofthese membrane polymers provided satisfactory performance when usedalone.

FIG. 20 shows an illustrative plot of the response of an electrodeovercoated with a bilayer membrane comprising a lower layer ofcrosslinked PVP (Polymer 2) and an upper layer of crosslinked Polymer 1Bto a 5 mM lactate solution. Unlike either Polymer 1B or PVP crosslinkedwith the same crosslinking agent (Polymer 2), a bilayer membranecomprising these membrane polymers surprisingly afforded a stablecurrent response over time at an acceptable level of sensitivity, eventhough neither polymer provided acceptable performance alone. Theresponse data in FIG. 20 was for an electrode dipped twice inFormulation 3 (Polymer 2) and four times in Formulation 2 (Polymer 1B).

The amount (thickness) of each membrane polymer in the bilayer membranemay vary the sensor performance, as shown hereinafter for Polymer 2 andPolymer 1A. Thus, the Gly3-crosslinked variant of Polymer 1B (i.e.,Polymer 1A) may similarly provide acceptable performance when combinedin a bilayer membrane with Polymer 2, even though neither polymerprovided acceptable performance alone.

FIG. 21 shows an illustrative plot of the response of an electrodeovercoated with a bilayer membrane comprising a lower layer ofcrosslinked PVP (Polymer 2) and an upper layer of crosslinked Polymer 1Ato a 5 mM lactate solution, in which the electrode was dip coated avariable number of times with Formulation 1 (Polymer 1A) and Formulation3 (Polymer 2). The crosslinker for PVP (Polymer 2) in this case remainedPEGDGE1000, but the crosslinker for Polymer 1A was Gly3, which showsthat this crosslinker can also be suitable for use in a bilayer membraneconfiguration. As shown in FIG. 21, dip coating the electrode twice inFormulation 3 and four times in Formulation 1 afforded a good balance ofsensitivity and a stable current response. Altering the number of dipcoating operations changed the thickness of each component of thebilayer membrane, as well as the mass ratio of the membrane polymers toeach other. As shown in FIG. 21, if the PVP layer is too thin (0 or 1Polymer 2 dips), the sensitivity is high but the response stability ispoor, whereas if it is too thick (3 or more dips), the electrodeexhibits low sensitivity and poor response stability in some cases.

Admixed Membrane Comprising Membrane Polymers 1B and 3: A combinedmembrane formulation (Formulation 6) was prepared by mixing 1.5 mL ofPVP in 80:20 ethanol:HEPES buffer (100 mg/mL), 2.5 mL of the copolymerused to prepare Formulations 1A and 1B in 80:20 ethanol:HEPES buffer(140 mg/mL), 0.175 mL of PEGDGE400 in 80:20 ethanol:HEPES buffer (100mg/mL), and 0.0132 mL of PDMS in ethanol (100 mg/mL). Thus, aftercrosslinking Formulation 6 contained Polymer 1B and Polymer 3, each ofwhich is crosslinked with PEG400.

FIG. 22 shows an illustrative plot of the response of an electrodeovercoated with an admixed membrane comprising crosslinked PVP (Polymer3) and crosslinked Polymer 1B to a 5 mM lactate solution. Like a bilayermembrane containing one of the same components (Polymer 1B), the admixedmembrane afforded a stable current response over time and an acceptablelevel of sensitivity. Moreover, the current responded rapidly andachieved a stable value as increasing amounts of lactate were added in 1mM increments (FIG. 23).

FIG. 24 shows an illustrative plot of the response a sensor overcoatedwith an admixed membrane comprising various ratios of crosslinked PVP(Polymer 3) and crosslinked Polymer 1B. As shown in FIG. 24, higheramounts of Polymer 1B increased the sensitivity but afforded poorerresponse stability.

Example 4: Performance of a Sensor Comprising Two Working ElectrodesOvercoated with a Bilayer Mass Transport Limiting Membrane. For thisexample, a first working electrode containing glucose oxidase and asecond working electrode containing lactate oxidase were overcoated witha bilayer membrane. The active area containing glucose oxidase wasdeposited using the glucose oxidase formulation as described in Example1 (Table 1). The active area containing lactate oxidase was depositedusing the lactate oxidase formulation as described in Example 3 (Table6). Active area deposition and curing was performed as described inExample 1, except for depositing five spots each having an area of 0.01mm² instead of the single spot having an area of 0.1 mm² in Example 1.Membrane polymer formulations corresponding to Formulation 2 (Polymer1B) and Formulation 4 (Polymer 3) from Example 3 were employed fordepositing the bilayer membrane in this example. Namely, Polymer 3 wasdeposited upon the second working electrode featuring lactate oxidase.Selective deposition upon the second working electrode was accomplishedby a modified slot coating procedure. Curing was then performed for 24hours at 25° C. Thereafter, the entire assembly (i.e., both workingelectrodes, the PVP coating upon the second working electrode, and thecounter and reference electrodes) was dip coated in Formulation 2.Curing was again performed for 24 hours at 25° C., followed by baking at56° C. in a desiccated environment for 48 hours. Thus, a homogeneousmembrane was deposited upon the first working electrode(glucose-responsive) and a bilayer membrane was deposited upon thesecond working electrode (lactate-responsive). The crosslinked PVP(Polymer 3) was in contact with the lactate-responsive active area uponthe second working electrode.

The sensor was used to assay for glucose and lactate simultaneously in100 mM PBS at 37° C. In a first experiment, the sensor was exposed for 2weeks at 37° C. to a 100 mM PBS solution containing 30 mM glucose and 5mM lactate. The sensor was held at +40 mV relative to Ag/AgCl for thistest. FIG. 25 shows an illustrative plot of the sensor response for eachworking electrode upon exposure to 30 mM glucose and 5 mM lactate. Asshown, the sensor response remained very steady over the observationperiod.

Next, glucose and lactate were added incrementally to 100 mM PBS at 37°C. to determine the responsiveness of the sensor toward each analyte.The sensor was again held at +40 mV relative to Ag/AgCl for this test.Glucose was added over a concentration range of 0-30 mM, and lactate wasadded over a concentration range of 0-5 mM. FIG. 26 shows anillustrative plot of the sensor response to varying concentrations ofglucose and lactate. As shown in FIG. 26, the sensor response was rapidfor both analytes and remained stable at a given analyte concentration.

Example 5: Detection of Ketones Using an Analyte Sensor Having InConcert Interacting Diaphorase and β-Hydroxybutyrate Dehydrogenase. Forthis example, the membrane formulation shown in Table 7 below was coatedonto a carbon working electrode. Deposition was performed to place sixspots, each having an area of around 0.01 mm², upon the workingelectrode. Following deposition, the working electrode was curedovernight at 25° C. Thereafter, a PVP membrane was applied to theworking electrode via dip coating using a coating solution formulatedwith 4 mL of 100 mg/mL PVP, 0.2 mL of 100 mg/mL PEGDGE400, and 0.0132 mLof 100 mg/mL PDMS. Membrane curing was performed for 24 hours at 25° C.,followed by 48 hours at 56° C. in desiccated vials.

TABLE 7 β-Hydroxybutyrate Dehydrogenase (HBDH) in 10 mM MES Buffer at pH= 5.5 Component Concentration (mg/mL) HBDH 8 Diaphorase 4 Albumin 8 NAD⁺8 Formula 1 Polymer 8 PEGDGE400 4

Ketone analyses were conducted by immersing the electrode in 100 mM PBSbuffer (pH=7.4) at 33° C. and introducing various amounts ofβ-hydroxybutyrate (0, 1, 2, 3, 4, 6 and 8 mM total β-hydroxybutyrateaddition). FIG. 27 shows four replicates of the response for anelectrode containing diaphorase, NAD⁺, and β-hydroxybutyratedehydrogenase when exposed to varying β-hydroxybutyrate concentrations.As shown, the current response increased over the course of severalminutes following exposure to a new β-hydroxybutyrate concentrationbefore stabilizing thereafter. FIG. 28 shows an illustrative plot ofaverage current response versus β-hydroxybutyrate concentration for theelectrodes of FIG. 27. The ketone sensors also exhibited a stableresponse over extended measurement times, as shown in FIG. 29. FIG. 29shows an illustrative plot of current response for the electrodes ofFIG. 27 when exposed to 8 mM of β-hydroxybutyrate in 100 mM PBS at 33°C. for 2 weeks. The mean signal loss over the measurement period wasonly 3.1%.

Example 6: Comparison of Lactate Sensor Response for Various SensorConfigurations. Two different lactate oxidase/polymer formulations foractive area deposition and two different membrane polymer formulationsfor mass transport limiting membrane deposition were prepared to assaythe performance of lactate-responsive sensors featuring variouspermutations of these formulations. Formulation details and the processused for preparing the analyte sensors are provided below. In general,the analyte sensors were prepared in a manner similar to that describedabove.

TABLE 8 Lactate Oxidase (LOX) in 10 mM HEPES Buffer at pH = 8(Formulation A) Component Concentration (mg/mL) LOX 24.6 Formula 1Polymer 20.4 PEGDGE400 7.5

TABLE 9 Lactate Oxidase (LOX) in 10 mM MES Buffer at pH = 5.5(Formulation B) Component Concentration (mg/mL) LOX 24.6 Human SerumAlbumin 24.6 Formula 1 Polymer 9.2 PEGDGE400 6.2

To deposit each active area, ˜20 nL of each solution was deposited upona carbon working electrode to form 6 discrete spots, each having an areaof approximately 0.01 mm². Formulation A was dispensed 4 times andFormulation B was dispensed 6 times to form the spots. Followingdeposition, the working electrode was cured overnight at 25° C.Formulation A corresponds to that used for depositing the active area ofglucose-responsive analyte sensors, except substituting lactate oxidasefor glucose oxidase.

Formulations for Mass Transport Limiting Membrane Deposition: Membranepolymer formulations were prepared in aqueous solution formulationsspecified in Tables 10 and 11 below.

TABLE 10 Polyvinylpyridine-co-Styrene Formulation in 80:20 Ethanol:HEPESBuffer-Gly3 Crosslinked (Formulation C) Component Concentration (mg/mL)Polyvinylpyridine-co-styrene polymer 111.7 Gly3 crosslinker 7.0polydimethylsiloxane 0.3

TABLE 11 Polyvinylpyridine Formulation in 80:20 Ethanol:HEPESBuffer-PEGDGE400 Crosslinked (Formulation D) Component Concentration(mg/mL) Polyvinylpyridine 94.6 PEGDGE400 crosslinker 5.1polydimethylsiloxane 0.3Dip coating was used to deposit a mass transport limiting membrane uponeach active area prepared as above. Formulation C was deposited using 4dips, and Formulation D was deposited using 4 dips. A wait time of about10 minutes between dips was used. Following the completion of dipcoating, the membranes were cured for 24 hours at 25° C., followed by 48hours at 56° C. in desiccated vials. Spray coating, screen printing, orsimilar processes may be alternately used to deposit the mass transportlimiting membrane. Formulation C corresponds to that used for depositinga mass transport limiting membrane within glucose-responsive analytesensors.

Lactate-responsive analyte sensors were prepared using the depositionconditions specified above. All possible combinations of active area andmass transport limiting membrane were prepared, with 8 sensors beingfabricated for each possible combination. After fabrication, each sensorwas exposed to a 5 mM lactate solution in 100 mM phosphate bufferedsaline (PBS) at 37° C. for 190 hours, with the working potential beingheld at +40 mV relative to Ag/AgCl. The tested combinations of activeareas and mass transport limiting membranes are specified in Table 12.Testing results are shown in FIG. 30.

TABLE 12 Sensor Mass Transport Group Active Area Limiting MembraneResult 1 Formulation A Formulation C Poor sensitivity 2 Formulation AFormulation D Poor sensitivity 3 Formulation B Formulation C Variablesensitivity, decreasing signal intensity over time 4 Formulation BFormulation D High sensitivity, stable signal intensity over time

As shown in FIG. 30, lactate-responsive analyte sensors having activeareas and mass transport limiting membranes formulated similarly tothose used successfully in glucose-responsive analyte sensors (Group 1)afforded poor performance when exposed to lactate. As shown, the signalintensity was under 0.5 nA for all of the tested samples, which isundesirably low for a viable lactate-responsive sensor. The signalintensity was even poorer when polyvinylpyridine and a differentcrosslinking agent were substituted for the polyvinylpyridine-co-styreneand Gly3 crosslinker in Formulation C (Group 2).

Incorporation of human serum albumin considerably improved the sensorperformance, as further shown in FIG. 30. Sample Group 3, for example,exhibited considerably higher signal intensities than were realized forany of the Group 1 or Group 2 samples. However, there was considerablyvariability in the initial signal intensity among this group of samples(>4 nA variance). Moreover, there was a steady decrease in the signalintensity from the initially observed maximum signal intensity. Theresponse variability and the poor signal stability over time likewisemakes the combination of this sample group unlikely to be suitable for aviable lactate-responsive analyte sensor.

Surprisingly, the combination of a human serum albumin-containing activearea and a mass transport limiting membrane comprising crosslinkedpolyvinylpyridine homopolymer (Group 4) produced an acceptablecombination of high signal intensity and extended signal stability overtime. As shown in FIG. 30, all of the replicate sensors of Group 4 hadinitial signal intensities clustered within 1 nA of each other between 4nA and 5 nA. This level of signal intensity and variability is withinthe range over which a commercially viable lactate-responsive analytesensor might be developed. Moreover, the signal intensity only varied afew tenths of a nA or less over 190 hours of signal observation, whichis again within a range that may be suitable for development of acommercially viable sensor.

As shown in FIG. 31, the observed current for the Group 4 sensorsresponded rapidly and achieved a stable value as increasing amounts oflactate were added in 1 mM increments to a PBS solution initially notcontaining lactate.

Unless otherwise indicated, all numbers expressing quantities and thelike in the present specification and associated claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the embodiments of the present invention. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claim, each numericalparameter should at least be construed in light of the number ofreported significant digits and by applying ordinary roundingtechniques.

Analyte Sensor Ignition Lock

Vehicle fail safes, such as ignition locks, are sometimes used toprevent an operator from operating a vehicle when impaired or otherwisenot in a condition to safely operate the vehicle. Operating the vehiclewhile impaired could potentially present significant dangers to theoperator and the public. One common type of ignition lock is designed toprevent drunk driving and, more specifically, to prevent individualsfrom operating a vehicle while intoxicated through alcohol use. Suchlock devices connect a breath-alcohol analyzer or optical sensor to thevehicle's ignition system, and the driver must successfully pass a bloodalcohol level test before the vehicle can be started.

Intoxication is one type of impairment or condition that an operator mayexperience that renders the operator unfit or unable to operate avehicle. However, other impairments and conditions can also afflict anoperator and should also be monitored closely to ensure the operatordoes not operate a vehicle while impaired. For example, an operator withdiabetes and driving while hypoglycemic (i.e., low blood sugar) couldpotentially undergo light-headedness, confusion, headache, loss ofconsciousness, seizures, and delayed reflexes, any of which couldendanger his/her own life and those in the vehicle or in the vicinity ofthe vehicle.

Analyte monitoring systems, have been developed to facilitate long-termmonitoring of analytes in bodily fluid (e.g., blood). Some analytemonitoring systems are designed to detect and monitor levels of bloodglucose, which can be helpful in treating diabetic conditions. Otheranalyte monitoring systems, however, are designed to detect and monitorother analytes present in an operator's bodily fluid, and abnormalanalyte levels detected in an operator may be indicative that theoperator is currently unfit to safely operate a vehicle.

The following discussion describes an analyte monitoring and vehiclecontrol system used to prevent operation of a vehicle when operatoranalyte levels cross a predetermined threshold. Having the sensorcontrol device 102 (FIG. 1) properly deployed allows a user tointelligently track and monitor bodily fluid analyte levels and trends.When some analyte levels surpass certain thresholds, physical orcognitive impairment may ensue that renders a user unfit to safelyoperate a vehicle. In such instances, the user should take appropriateaction to bring analyte levels back into safe ranges prior to attemptingto operate a vehicle. In some cases, however, a user may feel perfectlyfine to operate a vehicle but nonetheless have unsafe analyte levelsthat could suddenly trigger the onset of a dangerous physicalimpairment. In such cases, it may be advantageous to have a failsafesystem in place that prevents or warns the user from operating a vehicleand potentially placing self and/or others in danger.

FIG. 32 is a schematic diagram of an example analyte monitoring andvehicle control system 3200, according to one or more embodiments of thepresent disclosure. As illustrated, the analyte monitoring and vehiclecontrol system 3200 (hereafter “the system 3200) includes the sensorcontrol device 102, which may be deployed on a user or “operator” 3202and otherwise delivered to a target monitoring location on the body ofthe operator 3202, such as the back of an arm. As discussed above, thesensor control device 102 includes the sensor 104 (FIG. 1), and whenproperly deployed, the sensor 104 is positioned transcutaneously withinthe skin to detect and monitor analytes present within a bodily fluid ofthe operator 3202. The adhesive patch 105 (FIG. 1) applied to the bottomof the sensor control device 102 adheres to the skin to secure thesensor control device 102 in place during operation.

While the system 3200 is described herein as including the on-bodysensor control device 102 to detect and report analyte levels, thesystem 3200 may alternatively incorporate an ex vivo analyte sensor(e.g., a self-monitoring blood glucose “SMBG” meter), without departingfrom the scope of the disclosure. Accordingly, the term “sensor controldevice” should be interpreted herein to include not only on-body sensorsystems, as generally described above, but also traditional, hand-heldsensor systems.

As illustrated, the system 3200 may further include the reader device120, and the sensor control device 102 may be in communication with thereader device 120 via a local communication path or link to provideanalyte concentration data automatically, periodically, or as desired bythe operator 3202. The reader device 120 may be in communication with acontrol module 3204, which is in communication with the electricalsystem of a vehicle 3206 and powered by the vehicle battery or otherwisepowered by a separate battery. In such embodiments, data transmitted tothe reader device 120 from the sensor control device 102 may besubsequently transmitted by the reader device 120 to the control module3204 for processing. In other embodiments, however, the sensor controldevice 102 may communicate directly with the control module 3204 via anywireless communication protocol, such as BLUETOOTH®. In suchembodiments, the reader device 120 may or may not be necessary in thesystem 3200.

In the illustrated embodiment, the vehicle 3206 is depicted as anautomobile. As used herein, however, the term “vehicle” is used broadlyand is meant to include any kind of transportation vehicle that can beoperated by a human user or “operator,” but can also include autonomousvehicles used to transport humans. Examples of the vehicle 3206 include,but are not limited to, any type of automobile, truck, sport utilityvehicle, aircraft, watercraft, spacecraft, and or any other means oftransportation, or combinations thereof.

The control module 3204 may include a communications interface tocommunicate information to/from the sensor control device 102 and/or thereader device 120. In the case of an exemplary BLUETOOTH®-enabled sensorcontrol device 102 and/or reader device 120, a pairing mode may beentered into when the sensor control device 102 approaches the vehicle3206. Upon pairing, the control module 3204 may be programmed andconfigured to automatically detect the presence of and establishcommunication with the sensor control device 102 and/or the readerdevice 120. For example, when the operator 3202 approaches or enters thevehicle 3206, the control module 3204 may automatically detect thepresence of the sensor control device 102 and enable communicationtherebetween or with the reader device 120.

In some embodiments, the control module 3204 may be in communicationwith a vehicle user interface 3208 included in the vehicle 3206, such asan infotainment system, a touchscreen display, or an informationdisplay. In such embodiments, the control module 3204 may visuallycommunicate with the operator 3202 via the vehicle user interface 3208and may also be able to audibly communicate with the operator 3202 viathe audio speakers included in the vehicle 3206. In other embodiments,however, the control module 3204 may be configured to communicate withthe reader device 120 to be able to communicate with the operator 3202.

As illustrated, the control module 3204 may be or otherwise include acomputer system 3210 configured and otherwise programmed to controlvarious operations and/or systems of the vehicle 3206 based on real-timemeasured analyte levels of the operator 3202 as obtained by the sensorcontrol device 102. Operation of the vehicle 3206 is controlled,disabled, or modified by either disabling one or more critical systemsof the vehicle 3206 or by activating warning systems in the vehicle3206. When the real-time measured analyte levels of the operator 3202are within a predetermined safe range, then it may be considered safefor the operator 3202 to operate the vehicle 3206. When the real-timemeasured analyte levels of the operator 3202 fall outside thepredetermined safe range or cross a predetermined threshold, however,the computer system 3210 may then be programmed to control, disable, ormodify operation of the vehicle 3206.

In some embodiments, for example, the computer system 3210 may beconfigured to disable various critical vehicle systems when detectedanalyte levels of the operator 3202 fall outside of a predeterminedrange or otherwise cross a predetermined threshold, thus progressivelyand safely disabling operation of the vehicle when identifying theoperator 3202 as impaired for safe operation of the vehicle 3206.Critical vehicle systems of the vehicle 3206 that may be disabledinclude the ignition system (e.g., energy switching/control system), thetransmission system (or gear box), the fuel system, energy supply system(e.g., a battery, capacitor, conversion/reaction cell, etc.). Whenelevated or lowered (unsafe) analyte levels are detected, the computersystem 3210 may prevent the critical vehicle systems from functioning oroperating. Consequently, the operator 3202 will be unable to start oroperate the vehicle 3206, thereby preventing the operator 3202 fromplacing themselves and/or others in danger.

In other embodiments, or in addition thereto, the computer system 3210may be configured to activate various non-critical vehicle systems whendetected analyte levels of the operator 3202 surpass or cross apredetermined threshold. Non-critical vehicle systems that may beactivated include, for example, the vehicle horn, the vehicle lights, oran audible warning system installed in the vehicle 3206. In suchembodiments, activation of the non-critical vehicle systems may alertlaw enforcement and others (e.g., operators of adjacent vehicles,bystanders, pedestrians, etc.) of an operator 3202 that may be drivingin an impaired condition, thus allowing law enforcement to quicklyaddress any issues related thereto and placing others on notice of apotentially dangerous situation.

In yet other embodiments, or in addition thereto, the computer system3210 may be configured to automatically place a phone call to one ormore emergency contacts when analyte levels of the operator 3202 falloutside of a predetermined safe operating range or otherwise cross apredetermined threshold. In such embodiments, the computer system 3210may operate through the reader device 120 (e.g., a cellular phone) or acellular or satellite communication system incorporated into the vehicle3206 (e.g., OnStar®). In other embodiments, or in addition thereto, thecomputer system 3210 may be configured to automatically send a message(e.g., text or SMS message, email, etc.) to an emergency contact whenanalyte levels of the operator 3202 fall outside of a predetermined safeoperating range or otherwise cross a predetermined threshold. Exampleemergency contacts include, but are not limited to, a spouse, a parent,medical personnel (e.g., a doctor), a hospital, 911, or any combinationthereof.

In some embodiments, the system 3200 may further include one or moreproximity sensors 3212 configured to detect the presence of the operator3202 and, more particularly, the sensor control device 102. In suchembodiments, the proximity sensor(s) 3212 may be configured to monitorthe general area of the driver's seat 3214 within the vehicle 3206. Ifthe sensor control device 102 is detected within the area of thedriver's seat 3214 by the proximity sensor(s) 3212, that may provide apositive indication that the operator 3202 is in the driver's seat 3214and potentially attempting to operate the vehicle 3206. In such cases, asignal may be sent to the control module 3204 alerting the computersystem 3210 that the operator 3202 is in the vehicle 3206 andpotentially attempting to operate the vehicle 3206. If the real-timemeasured analyte levels of the operator 3202 are within a predeterminedsafe range or below a predetermined level, then the computer system 3210may allow the operator 3202 to operate the vehicle 3206. When thereal-time measured analyte levels of the operator 3202 fall outside thepredetermined safe range or cross a predetermined threshold, however,the computer system 3210 may control, disable, or modify operation ofthe vehicle 3206, as generally described above. As will be appreciated,the proximity sensor(s) 3212 may be advantageous in preventing operationof the vehicle 3206 only when the impaired operator 3202 is in thedriver's seat 3214 and ready to operate the vehicle 3206. Consequently,a user wearing the sensor control device 102 is able to ride as apassenger in the vehicle 3206 in any state without affecting operationof the control module 3204 or the vehicle 3206.

In some embodiments, the control module 3204 may further include avehicle status detection module 3216 configured to detect the currentstatus of the vehicle 3206, including whether the vehicle 3206 iscurrently moving or is stationary. In addition, the vehicle statusdetection module 3216 may be configured to determine whether or not themotor in the vehicle 3206 is currently operating or is stopped. In oneor more embodiments, the vehicle status detection module 3216 mayprovide a status signal to the control module 3204, and the controlmodule 3204 can then use the status signal to determine what vehicleoperations should be activated or disabled when the real-time measuredanalyte levels of the operator 3202 fall outside the predetermined saferange or cross a predetermined threshold. For example, when the statussignal indicates that the vehicle 3206 is stationary, the control module3204 can disable the vehicle fuel system, transmission system, ignitionsystem, or any combination thereof. In contrast, when the status signalindicates that the vehicle 3206 is moving, the control module 3204 canactivate the vehicle horn, flash the vehicle lights, or activate anaudible warning to the operator 3202 and/or those around the operator3202 that the operator 3202 is impaired.

In some embodiments, once the operator 3202 enters the vehicle 3206 orwhen the control module 3204 pairs with the sensor control device 102and/or the reader device 120, an app may be launched on the readerdevice 120 or the vehicle user interface 3208, and a digital dashboardmay appear on the reader device 120 and/or the vehicle user interface3208 that depicts current analyte levels, trend, historical data, andprojected analyte levels. If the current analyte levels fall outside ofa predetermined safe operating range, however, the computer system 3210may be programmed to disable one or more critical vehicle systems toprevent the operator 3202 from operating the vehicle 3206. In suchembodiments, a visual or audible alert may be issued by the controlmodule 3204 to inform the operator 3202 as to why the vehicle 3206 isnot starting. More particularly, a visual alert (e.g., a writtenmessage) may be generated and displayed on the reader device 120 or thevehicle user interface 3208, or an audible alert (e.g., a vocal message)may be transmitted through the speakers in the reader device 120 or thevehicle 3206.

If not done automatically, the operator 3202 may be prompted to obtain acurrent analyte level upon pairing the sensor control device 102 withthe control module 3204. In some cases, the vehicle 3206 may beprevented from being operated until a current analyte level is obtained.If the current analyte levels are within safe limits, the computersystem 3210 may allow operation of the vehicle 3206. In some aspects,and unless done automatically, the control module 3204 may prompt theoperator 3202 to obtain additional current analyte levels afteroperating the vehicle 3206 for a predetermined period of time (e.g.,after 1 hour, 2 hours, 5 hours, etc.).

In some embodiments, the control module 3204 may be configured to issuevisual or audible recommendations or coaching to the operator 3202 thatmay help bring measured analyte levels back into safe ranges. In suchembodiments, such visual or audible recommendations may prompt the userto take some action that could result in bringing analyte levels backinto safe ranges. Moreover, in some embodiments, the operator 3202 maybe able to communicate with the control module 3204 verbally by issuingverbal responses or commands. This may prove advantageous in helpingprevent distracted operation of the vehicle 3206.

In some embodiments, settings of the control module 3204 may becustomized by the operator 3202 to allow the user to make informeddecisions once unsafe analyte levels have been detected and a visual oraudible alert has been issued by the control module 3204. Morespecifically, in at least one embodiment, the control module 3204 mayinclude a bypass feature that the operator 3202 might enable to allowthe operator 3202 to operate the vehicle 3206 even when unsafe analytelevels have been measured. In such embodiments, the operator 3202 mayoperate the vehicle 3206 by acknowledging that the operator 3202 mightbe operating the vehicle 3206 in an impaired or unsafe health state.

In some embodiments, the computer system 3210 may be configured orotherwise programmed to calculate a predicted timeline when analytelevels of the operator 3202 may depart from a predetermined safe rangeor otherwise cross a predetermined threshold. In such embodiments, thecontrol module 3204 may be configured to issue visual or audible alertsto the operator 3202 indicating approximately how much time the operator3202 has before unsafe analyte levels may be reached and a potentialunsafe medical condition may ensue. Multiple alerts may be provided toindicate when the operator has specific time increments remaining beforeunsafe analyte levels are reached. For example, visual or audible alertsmay be issued when unsafe analyte levels will be reached within an hour,within a half hour, within 10 minutes, within 5 minutes, within 1minute, and any time increment therebetween. Furthermore, a visual oraudible alert may be issued once the analyte levels of the operatorreach an unsafe level or cross a predetermined threshold.

In some embodiments, if unsafe analyte levels are measured while theoperator 3202 is operating the vehicle 3206, the control module 3204 maybe configured to issue one or more alerts (visual or audible) warningthe operator 3202 of the unsafe analyte levels. In some cases, thevolume of the stereo in the vehicle 3206 may be automatically lowered toenable the operator 3202 to hear an audible alert. In such embodiments,the control module 3204 may be configured to suggest one or morecorrective actions to the operator 3202. Example corrective actionsinclude, but are not limited to, slowing and stopping the vehicle 3206,locating and driving to a nearby convenience store or pharmacy, andlocating a nearby hospital or medical facility. If the vehicle 3206 isan autonomous vehicle, and the current analyte levels place the operator3202 in potentially dangerous conditions, the control module 3204 mayautomatically direct the vehicle 3206 to a medical facility fortreatment. Alternatively, or in addition thereto, the control module3204 may progressively reduce or restrict the speed of the vehicle 3206when unsafe analyte levels are detected, thus forcing the operator 3202to come to a stop and remedy the issue before continuing to operate thevehicle 3206.

The system 3200 may be useful in several different scenarios to protectthe operator 3202 and/or those around the operator 3202 while driving.In some applications, the system 3200 may be incorporated voluntarily bythe operator to detect impairment in real-time. In other applications,the system 3200 may be required by the owner of the vehicle 3206 todetect impairment of the operator 3202. In such applications, the ownerof the vehicle 3206 may be a transport or trucking company. In yet otherapplications, the system 3200 may be legally imposed on the operator3202 to detect impairment.

Embodiments disclosed herein include:

K. An analyte monitoring and vehicle control system that includes asensor control device having a sensor that detects and monitors one ormore analytes present within a body of an operator, and a control modulein communication with the sensor control device and an electrical systemof a vehicle, the control module including a computer system programmedto receive and process data provided by the sensor control device,wherein operation of the vehicle is controlled or disabled by thecomputer system when a real-time measured analyte level of the operatorcrosses a predetermined safe threshold.

L. A method that includes detecting and monitoring one or more analytespresent within a body of an operator with a sensor control device havinga sensor, receiving and processes data provided by the sensor controldevice with a control module in communication with the sensor controldevice and an electrical system of a vehicle; and controlling ordisabling operation of the vehicle with a computer system of the controlmodule when a real-time measured analyte level of the operator crosses apredetermined safe threshold.

Each of embodiments K and L may have one or more of the followingadditional elements in any combination: Element 1: wherein the sensorcontrol device is coupled to the operator and the sensor istranscutaneously positioned beneath skin of the operator to detect andmonitor the analytes present within a bodily fluid of the operator.Element 2: wherein the sensor control device comprises an ex vivoanalyte sensor. Element 3: further comprising a reader device thatreceives the data from the sensor control device and transmits the datato the control module. Element 4: wherein the vehicle comprises atransportation vehicle selected from the group consisting of anautomobile, an autonomous vehicle, a truck, a sport utility vehicle, anaircraft, a watercraft, a spacecraft, or any combination thereof.Element 5: wherein sensor control device pairs with the control modulefor communication upon the operator approaching the vehicle. Element 6:further comprising a vehicle user interface included in the vehicle andin communication with the control module. Element 7: wherein operationof the vehicle is disabled by disabling one or more critical systems ofthe vehicle, the critical systems being selected from the groupconsisting of an ignition system, a transmission system, a fuel system,and an energy supply system. Element 8: wherein operation of the vehicleis controlled by at least one of activating one or more non-criticalsystems of the vehicle, calling or sending a message to one or moreemergency contacts, and progressively reducing a speed of the vehicle.Element 9: further comprising one or more proximity sensors installed onthe vehicle to monitor an area of a driver's seat of the vehicle anddetect a presence of the operator. Element 10: wherein the controlmodule further includes a vehicle status detection module that detectsthe current status of the vehicle. Element 11: wherein the controlmodule generates visual or audible alerts perceivable by the operatorwhen the real-time measured analyte level of the operator falls outsideof the predetermined safe threshold. Element 12: wherein the visual oraudible alerts are generated at specific time increments before unsafeanalyte levels are reached. Element 13: wherein the visual or audiblealerts comprise one or more suggested corrective actions communicated tothe operator. Element 14: wherein the control module includes a bypassfeature allowing the operator to operate the vehicle when the real-timemeasured analyte level of the operator crosses the predeterminedthreshold.

Element 15: further comprising receiving the data from the sensorcontrol device and transmitting the data to the control module with areader device in communication with the sensor control device and thecontrol module. Element 16: wherein disabling operation of the vehiclecomprises disabling one or more critical systems of the vehicle, thecritical systems being selected from the group consisting of an ignitionsystem, a transmission system, a fuel system, and an energy supplysystem. Element 17: wherein controlling operation of the vehiclecomprises at least one of activating one or more non-critical systems ofthe vehicle, calling or sending a message to one or more emergencycontacts, and progressively reducing a speed of the vehicle. Element 18:further comprising monitoring an area of a driver's seat of the vehicleand detecting a presence of the operator with one or more proximitysensors installed on the vehicle. Element 19: further comprisingdetecting the current status of the vehicle with a vehicle statusdetection module included in the control module. Element 20: furthercomprising generating visual or audible alerts perceivable by theoperator with the control module when the real-time measured analytelevel of the operator crosses the predetermined threshold.

One or more illustrative embodiments incorporating various features arepresented herein. Not all features of a physical implementation aredescribed or shown in this application for the sake of clarity. It isunderstood that in the development of a physical embodimentincorporating the embodiments of the present invention, numerousimplementation-specific decisions must be made to achieve thedeveloper's goals, such as compliance with system-related,business-related, government-related and other constraints, which varyby implementation and from time to time. While a developer's effortsmight be time-consuming, such efforts would be, nevertheless, a routineundertaking for those of ordinary skill in the art and having benefit ofthis disclosure.

While various systems, tools and methods are described herein in termsof “comprising” various components or steps, the systems, tools andmethods can also “consist essentially of” or “consist of” the variouscomponents and steps.

As used herein, the phrase “at least one of” preceding a series ofitems, with the terms “and” or “or” to separate any of the items,modifies the list as a whole, rather than each member of the list (i.e.,each item). The phrase “at least one of” allows a meaning that includesat least one of any one of the items, and/or at least one of anycombination of the items, and/or at least one of each of the items. Byway of example, the phrases “at least one of A, B, and C” or “at leastone of A, B, or C” each refer to only A, only B, or only C; anycombination of A, B, and C; and/or at least one of each of A, B, and C.

Therefore, the disclosed systems, tools and methods are well adapted toattain the ends and advantages mentioned as well as those that areinherent therein. The particular embodiments disclosed above areillustrative only, as the teachings of the present disclosure may bemodified and practiced in different but equivalent manners apparent tothose skilled in the art having the benefit of the teachings herein.Furthermore, no limitations are intended to the details of constructionor design herein shown, other than as described in the claims below. Itis therefore evident that the particular illustrative embodimentsdisclosed above may be altered, combined, or modified and all suchvariations are considered within the scope of the present disclosure.The systems, tools and methods illustratively disclosed herein maysuitably be practiced in the absence of any element that is notspecifically disclosed herein and/or any optional element disclosedherein. While systems, tools and methods are described in terms of“comprising,” “containing,” or “including” various components or steps,the systems, tools and methods can also “consist essentially of” or“consist of” the various components and steps. All numbers and rangesdisclosed above may vary by some amount. Whenever a numerical range witha lower limit and an upper limit is disclosed, any number and anyincluded range falling within the range is specifically disclosed. Inparticular, every range of values (of the form, “from about a to aboutb,” or, equivalently, “from approximately a to b,” or, equivalently,“from approximately a-b”) disclosed herein is to be understood to setforth every number and range encompassed within the broader range ofvalues. Also, the terms in the claims have their plain, ordinary meaningunless otherwise explicitly and clearly defined by the patentee.Moreover, the indefinite articles “a” or “an,” as used in the claims,are defined herein to mean one or more than one of the elements that itintroduces. If there is any conflict in the usages of a word or term inthis specification and one or more patent or other documents that may beincorporated herein by reference, the definitions that are consistentwith this specification should be adopted.

What is claimed is the following:
 1. An analyte sensor comprising: asensor tail configured for insertion into a tissue, the sensor tailcomprising at least a working electrode; and first and second activeareas disposed on the sensor tail, the first and second active areascomprising at least two different enzymes for measuring a concentrationof at least one analyte; wherein the first active area is overcoatedwith a first membrane polymer and a second membrane polymer that differfrom one another.
 2. The analyte sensor of claim 1, wherein the firstactive area is overcoated with an admixture of the first membranepolymer and the second membrane polymer, and one of the first membranepolymer and the second membrane polymer overcoats the second active areaas a homogenous membrane.
 3. The analyte sensor of claim 1, wherein thefirst active area is overcoated with a bilayer membrane comprising thefirst membrane polymer disposed upon the second membrane polymer, andthe second membrane polymer overcoats the second active area as ahomogenous membrane.
 4. The analyte sensor of claim 1, wherein the firstactive area comprises a first enzyme of the at least two differentenzymes and the second active area comprises a second enzyme of the atleast two different enzymes.
 5. The analyte sensor of claim 4, whereinfirst enzyme is unreactive with the at least one analyte, and the firstand second enzymes are capable of interacting in concert to generate asignal proportional to the concentration of the at least one analyte.potential of the first active area is sufficiently separated from theoxidation-reduction potential of the second active area to allowproduction of a signal from the first active area independent ofproduction of a signal from the second active area.
 13. The analytesensor of claim 12, wherein the oxidation-reduction potential of thefirst active area is separated from the oxidation-reduction potential ofthe second active area by at least about 100 mV.
 14. The analyte sensorof claim 12, wherein the signal from the first active area correspondsto the first analyte concentration and the signal from the second activearea corresponds to the second analyte concentration.
 15. The analytesensor of claim 12, wherein the first active area comprises a firstelectron transfer agent and the second active area comprises a secondelectron transfer agent different from the first electron transferagent.
 16. An analyte sensor comprising: a sensor tail configured forinsertion into a tissue and comprising at least a working electrode; andat least two active areas disposed on the sensor tail, each active areacomprising an enzyme, an electron transfer agent, and a polymer; whereinthe enzyme in each active area is different and responsive to differentanalytes; and wherein each active area has an oxidation-reductionpotential, and the oxidation-reduction potential of a first active areais sufficiently separated from the oxidation-reduction potential of asecond active area to allow production of a signal from the first activearea independent of production of a signal from the second active area.17. The analyte sensor of claim 16, wherein the oxidation-reductionpotential of the first active area is separated from theoxidation-reduction potential of the
 6. The analyte sensor of claim 5,wherein the second enzyme is capable of converting the at least oneanalyte into a product reactive with the first enzyme, such that thefirst enzyme is capable of reacting the product to generate a signal atthe working electrode.
 7. The analyte sensor of claim 5, wherein thefirst enzyme is xanthine oxidase and the second enzyme is glucoseoxidase, at least one of the first active area and the second activearea further comprising catalase.
 8. The analyte sensor of claim 7,wherein the catalase is present in the first active area.
 9. The analytesensor of claim 5, wherein the first active area is disposed directlyupon the working electrode and further comprises an electron transferagent.
 10. The analyte sensor of claim 5, wherein the first membranepolymer is disposed directly upon the first active area, the secondactive area is disposed directly upon the first membrane polymer, andthe second membrane polymer is disposed directly upon the second activearea.
 11. The analyte sensor of claim 4, wherein the sensor tailcomprises a first working electrode and a second working electrode, thefirst active area is disposed on a surface of the first workingelectrode, the second active area is disposed on a surface of the secondworking electrode, the first enzyme is reactive with a first analyte togenerate a signal proportional to the concentration the first analyte,and the second enzyme is reactive with a second analyte to generate asignal proportional to the concentration the second analyte.
 12. Theanalyte sensor of claim 4, wherein each of the first and second activeareas has an oxidation-reduction potential, and the oxidation-reductionsecond active area by at least about 100 mV.
 18. The analyte sensor ofclaim 16, wherein the first active area comprises a first electrontransfer agent and the second active area comprises a second electrontransfer agent different from the first electron transfer agent.
 19. Theanalyte sensor of claim 16, wherein the first and second active areasare overcoated with a mass transport limiting membrane, the first activearea being overcoated with a single membrane polymer and the secondactive area being overcoated with two or more different membranepolymers.
 20. The analyte sensor of claim 16, further comprising: acontrol module in communication with the analyte sensor and anelectrical system of a vehicle, the control module including a computersystem programmed to receive and process data provided by the analytesensor, wherein operation of the vehicle is controlled or disabled bythe computer system when a real-time measured analyte level of theoperator crosses a predetermined safe threshold.